Structured Review

Millipore pet28a
Digestion analysis of pJET1.2-CPA; A) pJET1.2-CPB; B) <t>PET28a-CPA;</t> C) and PET28a-CPB; D) with HindIII and BamH1. Band 1, 2:pJET1.2 (2964 bp ) and CPA (650 bp ), Band 3, 4: pJET1.2 (2964 bp ) and CPB (950 bp ), Band 5, 6: PET28a (5369 bp ) and CPB (950 bp ), and Band 7,8: PET28a (5369 bp ) and CPA (650 bp ).
Pet28a, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 804 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction"

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction

Journal: Avicenna Journal of Medical Biotechnology

doi:

Digestion analysis of pJET1.2-CPA; A) pJET1.2-CPB; B) PET28a-CPA; C) and PET28a-CPB; D) with HindIII and BamH1. Band 1, 2:pJET1.2 (2964 bp ) and CPA (650 bp ), Band 3, 4: pJET1.2 (2964 bp ) and CPB (950 bp ), Band 5, 6: PET28a (5369 bp ) and CPB (950 bp ), and Band 7,8: PET28a (5369 bp ) and CPA (650 bp ).
Figure Legend Snippet: Digestion analysis of pJET1.2-CPA; A) pJET1.2-CPB; B) PET28a-CPA; C) and PET28a-CPB; D) with HindIII and BamH1. Band 1, 2:pJET1.2 (2964 bp ) and CPA (650 bp ), Band 3, 4: pJET1.2 (2964 bp ) and CPB (950 bp ), Band 5, 6: PET28a (5369 bp ) and CPB (950 bp ), and Band 7,8: PET28a (5369 bp ) and CPA (650 bp ).

Techniques Used:

SDS PAGE for crude lyaste of E. coli transformed with the PET28a-CPA; A) and PET28a-CPB; B) Control: E. coli BL21 strain, lane 1: E. coli BL21 strain before induction at time 0, lane 2: E. coli BL21 strain one hour after induction, lane 3: E. coli BL21 strain two hours after induction, lane 4: E. coli BL21 strain three hours after induction, and lane 5: E. coli BL21 strain four hr after induction.
Figure Legend Snippet: SDS PAGE for crude lyaste of E. coli transformed with the PET28a-CPA; A) and PET28a-CPB; B) Control: E. coli BL21 strain, lane 1: E. coli BL21 strain before induction at time 0, lane 2: E. coli BL21 strain one hour after induction, lane 3: E. coli BL21 strain two hours after induction, lane 4: E. coli BL21 strain three hours after induction, and lane 5: E. coli BL21 strain four hr after induction.

Techniques Used: SDS Page, Transformation Assay

2) Product Images from "The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus"

Article Title: The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus

Journal:

doi:

Overproduction and purification of RheA. SDS/PAGE analysis of crude extracts from E. coli BL21λDE3 carrying pET28a (lane 2) or pOLP12B (lane 3) and purified RheA protein after Ni-NTA affinity chromatography (lane 4). Molecular mass standards (New England Biolabs) were loaded in lane 1.
Figure Legend Snippet: Overproduction and purification of RheA. SDS/PAGE analysis of crude extracts from E. coli BL21λDE3 carrying pET28a (lane 2) or pOLP12B (lane 3) and purified RheA protein after Ni-NTA affinity chromatography (lane 4). Molecular mass standards (New England Biolabs) were loaded in lane 1.

Techniques Used: Purification, SDS Page, Affinity Chromatography

3) Product Images from "Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao"

Article Title: Evaluation of the Allergenicity Potential of TcPR-10 Protein from Theobroma cacao

Journal: PLoS ONE

doi: 10.1371/journal.pone.0037969

SDS-PAGE (15%) analysis of recombinants TcPR-10 Wild Types (wt) and Mutant (mut) proteins expressed in E. coli BL21(DE3). 1 – Soluble fraction TcPR-10 mut; 2 – Insoluble fraction TcPR10 mut; 3 – Soluble fraction TcPR-10 wt; 4 – Insoluble fraction TcPR10 wt; 5 - pET28a-TcPR-10 mut without induction; 6 - pET28a-TcPR-10 mut after induction; 7 - pET28a-TcPR-10 wt without induction; 8 - pET28a-TcPR-10 mut 3 h after induction; M - Protein molecular weight markers.
Figure Legend Snippet: SDS-PAGE (15%) analysis of recombinants TcPR-10 Wild Types (wt) and Mutant (mut) proteins expressed in E. coli BL21(DE3). 1 – Soluble fraction TcPR-10 mut; 2 – Insoluble fraction TcPR10 mut; 3 – Soluble fraction TcPR-10 wt; 4 – Insoluble fraction TcPR10 wt; 5 - pET28a-TcPR-10 mut without induction; 6 - pET28a-TcPR-10 mut after induction; 7 - pET28a-TcPR-10 wt without induction; 8 - pET28a-TcPR-10 mut 3 h after induction; M - Protein molecular weight markers.

Techniques Used: SDS Page, Mutagenesis, Molecular Weight

4) Product Images from "Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri"

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00072

Mass spectra showing the AHL profile of spent culture supernatant of E. coli BL21(DE3)pLysS harboring pET28a_ cneI . ACN (A) was used as blank. The EIC spectra of E. coli with pET_28a alone (B) and with pET28a_ cneI (C) were compared with that of synthetic AHL, C4-HSL (D) at the same retention time. The detection of the peaks with m/z 172.100 signify the presence of C4-HSL as shown by arrows.
Figure Legend Snippet: Mass spectra showing the AHL profile of spent culture supernatant of E. coli BL21(DE3)pLysS harboring pET28a_ cneI . ACN (A) was used as blank. The EIC spectra of E. coli with pET_28a alone (B) and with pET28a_ cneI (C) were compared with that of synthetic AHL, C4-HSL (D) at the same retention time. The detection of the peaks with m/z 172.100 signify the presence of C4-HSL as shown by arrows.

Techniques Used: Positron Emission Tomography

The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of CneI overexpression (Lanes 1–5) and purification (Lanes 6–8) from E. coli BL21(DE3)pLysS. Lane 1: Cell lysate of E. coli BL21(DE3)pLysS. Lanes 2 and 3: Cell lysate of E. coli BL21(DE3)pLysS transformed with empty pET28a with and without IPTG induction, respectively. Lanes 4 and 5: Cell lysates of E. coli BL21(DE3)pLysS harboring pET28a_ cneI with and without induction, respectively. Lane 6: Flowthrough fraction. Lane 7: Wash fraction. Lane 8: Eluted fraction. Lane 9: PageRuler prestained protein ladder in kiloDalton (kDa). (Thermo Scientific, Waltham, MA, USA).
Figure Legend Snippet: The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) profile of CneI overexpression (Lanes 1–5) and purification (Lanes 6–8) from E. coli BL21(DE3)pLysS. Lane 1: Cell lysate of E. coli BL21(DE3)pLysS. Lanes 2 and 3: Cell lysate of E. coli BL21(DE3)pLysS transformed with empty pET28a with and without IPTG induction, respectively. Lanes 4 and 5: Cell lysates of E. coli BL21(DE3)pLysS harboring pET28a_ cneI with and without induction, respectively. Lane 6: Flowthrough fraction. Lane 7: Wash fraction. Lane 8: Eluted fraction. Lane 9: PageRuler prestained protein ladder in kiloDalton (kDa). (Thermo Scientific, Waltham, MA, USA).

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Over Expression, Purification, Transformation Assay

5) Product Images from "Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration"

Article Title: Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

Journal: PLoS ONE

doi: 10.1371/journal.pone.0006980

SDS-PAGE analysis of different samples taken during the purification process of esterase LipC. Lane 1: crude supernatant of pET28a-LipC non-induced culture; Lanes 2 and 6: crude extract of induced culture; Lane 3: purification on Ni-NTA affinity Sepharose column; Lane 4: further purification with DEAE column; Lanes 5 and 6: further purification with Sephadex-G200. Lanes 1 to 5 were stained with Coomassie Brilliant Blue and Lanes 6 was stained with α-naphtyl acetate and Fast Blue for the detection of hydrolase activity.
Figure Legend Snippet: SDS-PAGE analysis of different samples taken during the purification process of esterase LipC. Lane 1: crude supernatant of pET28a-LipC non-induced culture; Lanes 2 and 6: crude extract of induced culture; Lane 3: purification on Ni-NTA affinity Sepharose column; Lane 4: further purification with DEAE column; Lanes 5 and 6: further purification with Sephadex-G200. Lanes 1 to 5 were stained with Coomassie Brilliant Blue and Lanes 6 was stained with α-naphtyl acetate and Fast Blue for the detection of hydrolase activity.

Techniques Used: SDS Page, Purification, Staining, Activity Assay

6) Product Images from "Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product Glycodiversification"

Article Title: Engineering a Carbohydrate-processing Transglycosidase into Glycosyltransferase for Natural Product Glycodiversification

Journal: Scientific Reports

doi: 10.1038/srep21051

( A ) Scheme of the glycosylation reaction based on which the high-throughput screening method was developed. ( B ) The representative activity data of glycosylation of fluorescent 4-MU illustrating ~100 random members from the GTF-D saturation mutagenesis library screening. The wild-type enzyme and mutant M4 were indicated. Strain BL21(DE3) harboring plasmid pET28a was used as control. Activities were calculated as the fluorescence differences of the variants between 0 (before the reaction) and 5 h (after the reaction).
Figure Legend Snippet: ( A ) Scheme of the glycosylation reaction based on which the high-throughput screening method was developed. ( B ) The representative activity data of glycosylation of fluorescent 4-MU illustrating ~100 random members from the GTF-D saturation mutagenesis library screening. The wild-type enzyme and mutant M4 were indicated. Strain BL21(DE3) harboring plasmid pET28a was used as control. Activities were calculated as the fluorescence differences of the variants between 0 (before the reaction) and 5 h (after the reaction).

Techniques Used: High Throughput Screening Assay, Activity Assay, Mutagenesis, Library Screening, Plasmid Preparation, Fluorescence

7) Product Images from "Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene"

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2015.00240

Analysis of aciI gene and protein. (A) Ethidium bromide-stained agarose gel containing aciI (gene amplification by PCR). Lanes 1 and 2 shows the amplified 552 bp amplicon. 5 μl of PCR products were loaded into each lane and electrophoresis was performed at 100 V. (B) SDS-PAGE analysis of the purified recombinant AciI protein. Lane 3, cell lysates of non-induced E. coli BL21 harboring pET28a-aciI; Lane 4, cell lysates of induced E. coli BL21 harboring pET28a-aciI; lane 5, flow-through fraction of purification step; lane 6, wash fraction of purification step; lane 7, eluted fraction containing recombinant AciI protein; lane M1, 1 kb DNA marker (Fermentas, Thermo Fisher Scientific, USA); lane M2, molecular weight markers (Bio-Rad, USA) with mass of each marker protein in kDa as indicated. The same amount of protein was loaded into each lane and subjected to electrophoresis at 150 V.
Figure Legend Snippet: Analysis of aciI gene and protein. (A) Ethidium bromide-stained agarose gel containing aciI (gene amplification by PCR). Lanes 1 and 2 shows the amplified 552 bp amplicon. 5 μl of PCR products were loaded into each lane and electrophoresis was performed at 100 V. (B) SDS-PAGE analysis of the purified recombinant AciI protein. Lane 3, cell lysates of non-induced E. coli BL21 harboring pET28a-aciI; Lane 4, cell lysates of induced E. coli BL21 harboring pET28a-aciI; lane 5, flow-through fraction of purification step; lane 6, wash fraction of purification step; lane 7, eluted fraction containing recombinant AciI protein; lane M1, 1 kb DNA marker (Fermentas, Thermo Fisher Scientific, USA); lane M2, molecular weight markers (Bio-Rad, USA) with mass of each marker protein in kDa as indicated. The same amount of protein was loaded into each lane and subjected to electrophoresis at 150 V.

Techniques Used: Staining, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Electrophoresis, SDS Page, Purification, Recombinant, Flow Cytometry, Marker, Molecular Weight

Mass spectrometry (MS) analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a-aciI showing the presence of 3-oxo-C12-HSL and 3-hydroxy-C12-HSL. By comparing with the corresponding synthetic AHL standards, the mass spectra demonstrated the presence of (A) 3-oxo-C12-HSL at m/z 298.1000 and (B) 3-hydroxy-C12-HSL at m/z 300.1000. The retention time for 3-oxo-C12-HSL and 3-hydroxy-C12-HSL are 6.684 min and 5.867 min, respectively. (i) Mass spectra of E. coli BL21 harboring pET28a alone (control); (ii) mass spectra of non-induced E. coli BL21 harboring pET28a-aciI (control); (iii) mass spectra of induced E. coli BL21 harboring pET28a-aciI.
Figure Legend Snippet: Mass spectrometry (MS) analyses of the extract of spent culture supernatant from IPTG-induced E. coli BL21 harboring pET28a-aciI showing the presence of 3-oxo-C12-HSL and 3-hydroxy-C12-HSL. By comparing with the corresponding synthetic AHL standards, the mass spectra demonstrated the presence of (A) 3-oxo-C12-HSL at m/z 298.1000 and (B) 3-hydroxy-C12-HSL at m/z 300.1000. The retention time for 3-oxo-C12-HSL and 3-hydroxy-C12-HSL are 6.684 min and 5.867 min, respectively. (i) Mass spectra of E. coli BL21 harboring pET28a alone (control); (ii) mass spectra of non-induced E. coli BL21 harboring pET28a-aciI (control); (iii) mass spectra of induced E. coli BL21 harboring pET28a-aciI.

Techniques Used: Mass Spectrometry

8) Product Images from ""

Article Title:

Journal:

doi: 10.1074/jbc.M111.313304

35 S-SmSrpQ complex. a , SmSrpQ was cloned into pET28a+ and expressed using the rabbit reticulolysate system in the presence of [ 35 S]methionine yielding a protein of ∼47 kDa. When incubated with cercarial lysate (at 25 °C, 30 °C,
Figure Legend Snippet: 35 S-SmSrpQ complex. a , SmSrpQ was cloned into pET28a+ and expressed using the rabbit reticulolysate system in the presence of [ 35 S]methionine yielding a protein of ∼47 kDa. When incubated with cercarial lysate (at 25 °C, 30 °C,

Techniques Used: Clone Assay, Incubation

9) Product Images from "Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli"

Article Title: Identification of a heterologous cellulase and its N-terminus that can guide recombinant proteins out of Escherichia coli

Journal: Microbial Cell Factories

doi: 10.1186/s12934-015-0230-8

Cell lysis determination and inactive Cel-CD secretion analysis. (A) Immunoblotting detection of Cel-CD, GroEL and MBP in the culture medium. Samples were subjected to immunoblotting with an anti-Cel-CD antibody, an anti-GroEL antibody or an anti-MBP antibody. (B) SDS analysis of cellulase (wt) and its mutants (E160Q, E160Q E274Q) secreted into the culture medium. The activities of the secreted mutants of Cel-CD (E160Q, E160Q E274Q) were analyzed by Congo red staining method (4 h, 6 h, 8 h, 16 h). (C) TEM images of E. coli BL21 (DE3) that containing different plasmids. (1) pET28a/cel, (2) pET28a/E160Q, (3) pET28a/E160Q E274Q, (4) pET28a. Protein samples were separated on 12% SDS-PAGE. Aliquots corresponding to 20 μL of the culture medium were loaded onto the gel. Molecular size markers are shown in kDa. Lane M is the marker; Lane C is the control of collected cells.
Figure Legend Snippet: Cell lysis determination and inactive Cel-CD secretion analysis. (A) Immunoblotting detection of Cel-CD, GroEL and MBP in the culture medium. Samples were subjected to immunoblotting with an anti-Cel-CD antibody, an anti-GroEL antibody or an anti-MBP antibody. (B) SDS analysis of cellulase (wt) and its mutants (E160Q, E160Q E274Q) secreted into the culture medium. The activities of the secreted mutants of Cel-CD (E160Q, E160Q E274Q) were analyzed by Congo red staining method (4 h, 6 h, 8 h, 16 h). (C) TEM images of E. coli BL21 (DE3) that containing different plasmids. (1) pET28a/cel, (2) pET28a/E160Q, (3) pET28a/E160Q E274Q, (4) pET28a. Protein samples were separated on 12% SDS-PAGE. Aliquots corresponding to 20 μL of the culture medium were loaded onto the gel. Molecular size markers are shown in kDa. Lane M is the marker; Lane C is the control of collected cells.

Techniques Used: Lysis, Staining, Transmission Electron Microscopy, SDS Page, Marker

10) Product Images from "Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure"

Article Title: Response of a Mu-class glutathione S-transferase from black tiger shrimp Penaeus monodon to aflatoxin B1 exposure

Journal: SpringerPlus

doi: 10.1186/s40064-016-2381-4

Expression and purification of the recombination PmMuGST fusion protein. Equal amounts of proteins (30 μg) were subject to SDS-PAGE and western blotting analysis. a Protein samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane M , protein standard; lane 1 , crude extract of BL 21 (DE3) without plasmid; lane 2 , crude extract of the transformed BL21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 3 , purified PmMuGST fusion protein. b Protein samples were analyzed by immunoblotting with anti- PmMuGST antibody. Lane M , protein standard; lane 1 , crude extract of the transformed BL 21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 2 , purified PmMuGST fusion protein
Figure Legend Snippet: Expression and purification of the recombination PmMuGST fusion protein. Equal amounts of proteins (30 μg) were subject to SDS-PAGE and western blotting analysis. a Protein samples were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Lane M , protein standard; lane 1 , crude extract of BL 21 (DE3) without plasmid; lane 2 , crude extract of the transformed BL21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 3 , purified PmMuGST fusion protein. b Protein samples were analyzed by immunoblotting with anti- PmMuGST antibody. Lane M , protein standard; lane 1 , crude extract of the transformed BL 21 (DE3) with recombined pET28a (+) plasmid induced with IPTG; lane 2 , purified PmMuGST fusion protein

Techniques Used: Expressing, Purification, SDS Page, Western Blot, Staining, Plasmid Preparation, Transformation Assay

11) Product Images from "The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice"

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

Journal: BioMed Research International

doi: 10.1155/2013/241721

Diagramme of pET28a-6 × his-4 × T β 4. lac I, lac repressor coding gene; Kan: kanamycin coding gene; 6 × his, a DNA sequence encoding six histidines; 4 × Tβ 4, four copies of a DNA sequence which encode Tβ 4 arranged in tandem; origin, DNA sequence of pBR322 origin of replication; f1 origin, DNA sequence of phage f1 origin of replication.
Figure Legend Snippet: Diagramme of pET28a-6 × his-4 × T β 4. lac I, lac repressor coding gene; Kan: kanamycin coding gene; 6 × his, a DNA sequence encoding six histidines; 4 × Tβ 4, four copies of a DNA sequence which encode Tβ 4 arranged in tandem; origin, DNA sequence of pBR322 origin of replication; f1 origin, DNA sequence of phage f1 origin of replication.

Techniques Used: Sequencing

(a) Expression of recombinant 4 × T β 4 in BL21 cells at different induction times. Lane M, middle-molecular mass protein markers; lanes from 1 to 5: induced expression of the recombinant 4 × T β 4 by IPTG (1 mM) at 0, 2, 4, 6, and 8 hs, respectively; lane 6: induced BL21 containing pET28a by IPTG (1 mM) for 6 hs. Red arrows indicate specific band of E. coli -derived 6 × his-×T β 4. (b) Western blot analysis of the E. coli -derived 6 × his-4 × T β 4. Lane M: middle-molecular-mass protein markers; lanes from 1 to 5: BL21 harboring pET28a-6 × his-4 × T β 4 after IPTG (1 mM) induction at 37°C for 0, 2, 4, 6, and 8 h, respectively; lane 6: induction of expression of the BL21 containing pET28a by IPTG (1 mM) at 37°C for 6 h. Red arrows indicate the specific hybridization signal for the E. coli -derived 6 × his-×T β 4.
Figure Legend Snippet: (a) Expression of recombinant 4 × T β 4 in BL21 cells at different induction times. Lane M, middle-molecular mass protein markers; lanes from 1 to 5: induced expression of the recombinant 4 × T β 4 by IPTG (1 mM) at 0, 2, 4, 6, and 8 hs, respectively; lane 6: induced BL21 containing pET28a by IPTG (1 mM) for 6 hs. Red arrows indicate specific band of E. coli -derived 6 × his-×T β 4. (b) Western blot analysis of the E. coli -derived 6 × his-4 × T β 4. Lane M: middle-molecular-mass protein markers; lanes from 1 to 5: BL21 harboring pET28a-6 × his-4 × T β 4 after IPTG (1 mM) induction at 37°C for 0, 2, 4, 6, and 8 h, respectively; lane 6: induction of expression of the BL21 containing pET28a by IPTG (1 mM) at 37°C for 6 h. Red arrows indicate the specific hybridization signal for the E. coli -derived 6 × his-×T β 4.

Techniques Used: Expressing, Recombinant, Derivative Assay, Western Blot, Hybridization

12) Product Images from "High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development"

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-10-56

VP1 protein expression in E. coli using various expression vectors . The VP1 protein expression in E. coli BL21(DE3) using the expression vectors, pET28a (A) and pGEX-4T-1 (B) was analyzed by SDS-PAGE and Western-blotting. Anti-His tag and anti-GST tag monoclonal antibody were respectively used for the recognition of differently tagged VP1 proteins expressed by the different expression vectors used. Lane M, pre-stained protein marker; lane 1 and lane 3, before IPTG induction; lane 2 and 4, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.
Figure Legend Snippet: VP1 protein expression in E. coli using various expression vectors . The VP1 protein expression in E. coli BL21(DE3) using the expression vectors, pET28a (A) and pGEX-4T-1 (B) was analyzed by SDS-PAGE and Western-blotting. Anti-His tag and anti-GST tag monoclonal antibody were respectively used for the recognition of differently tagged VP1 proteins expressed by the different expression vectors used. Lane M, pre-stained protein marker; lane 1 and lane 3, before IPTG induction; lane 2 and 4, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.

Techniques Used: Expressing, SDS Page, Western Blot, Staining, Marker

Schematic diagram of the constructs used for CVA VP1 protein expression . (A) Schematic representation of the VP1 protein variants and expression vectors used in this study. The designations of the VP1 protein and its expression vectors are indicated, a, b and c. The first two constructs, a and b, contained the full-length VP1 gene cloned into vectors pET28a and pGEX-4T-1, for expression of VP1 protein with a six-histidine (6 × His) tag and glutathione-s-transferase (GST) tag at its N-terminus, respectively. Construct c contained VP1 gene with codon-optimized at its N-terminus; this was derived from construct b by replacing the rare codons in the area 1-321 bp without altering amino acid sequence. The N-terminal codon-optimized VP1 gene, opt-N, was fused with C-terminal domain of VP1 gene (VP1 C-term) by overlapping PCR, and then cloned into pGEX-4T-1. (B) Sequence comparison between the WT VP1 and Opt VP1 gene. The nucleotide sequences were compared between the original VP1 gene (WT VP1) and the sequence of codon-optimized VP1 gene (Opt VP1) over the N-terminal region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.
Figure Legend Snippet: Schematic diagram of the constructs used for CVA VP1 protein expression . (A) Schematic representation of the VP1 protein variants and expression vectors used in this study. The designations of the VP1 protein and its expression vectors are indicated, a, b and c. The first two constructs, a and b, contained the full-length VP1 gene cloned into vectors pET28a and pGEX-4T-1, for expression of VP1 protein with a six-histidine (6 × His) tag and glutathione-s-transferase (GST) tag at its N-terminus, respectively. Construct c contained VP1 gene with codon-optimized at its N-terminus; this was derived from construct b by replacing the rare codons in the area 1-321 bp without altering amino acid sequence. The N-terminal codon-optimized VP1 gene, opt-N, was fused with C-terminal domain of VP1 gene (VP1 C-term) by overlapping PCR, and then cloned into pGEX-4T-1. (B) Sequence comparison between the WT VP1 and Opt VP1 gene. The nucleotide sequences were compared between the original VP1 gene (WT VP1) and the sequence of codon-optimized VP1 gene (Opt VP1) over the N-terminal region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.

Techniques Used: Construct, Expressing, Clone Assay, Derivative Assay, Sequencing, Polymerase Chain Reaction

13) Product Images from "Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli"

Article Title: Recombinant L-Asparaginase from Zymomonas mobilis: A Potential New Antileukemic Agent Produced in Escherichia coli

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156692

(a) Cell growth and activity of L-asparaginase using E . coli BL21 (DE3)/pET26b/ ans (extracellular expression) induced after 203 minutes of culture; (b) growth and expression of the protein using E . coli BL21 (DE3)/pET28a/ ans (intracellular expression) induced after 198 minutes of culture; (c) production of acetic acid over time; (d) specific activity of L-asparaginase over time as of induction of protein expression using the recombinant strains of L-asparaginase ( E . coli BL21 (DE3)/pET26b/ ans and E . coli BL21 (DE3)/pET28a/ ans ). The results are the mean of the data, with error bars representing the standard deviation of triplicate values.
Figure Legend Snippet: (a) Cell growth and activity of L-asparaginase using E . coli BL21 (DE3)/pET26b/ ans (extracellular expression) induced after 203 minutes of culture; (b) growth and expression of the protein using E . coli BL21 (DE3)/pET28a/ ans (intracellular expression) induced after 198 minutes of culture; (c) production of acetic acid over time; (d) specific activity of L-asparaginase over time as of induction of protein expression using the recombinant strains of L-asparaginase ( E . coli BL21 (DE3)/pET26b/ ans and E . coli BL21 (DE3)/pET28a/ ans ). The results are the mean of the data, with error bars representing the standard deviation of triplicate values.

Techniques Used: Activity Assay, Expressing, Recombinant, Standard Deviation

14) Product Images from "ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains"

Article Title: ZBP1 subcellular localization and association with stress granules is controlled by its Z-DNA binding domains

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl575

Comparison of Zα domains ( A ) and schematic representation of constructs used for transfections ( B ) and protein expression ( C ). Comparison of Zα domains of human (hs) and mouse (mm) ZBP1, human ADAR1, zebrafish (dr) PKZ, vaccinia virus (vv) E3L and yaba-like disease virus (yldv) E3L is shown (A). The structures of the mouse (mm) Zα ZBP1 , human (hs) Zα ADAR1 , yaba-like disease virus (yldv) Zα E3L domains have been determined in complex with Z-DNA. Residues that make contact with Z-DNA, or the analogous residues in other Zα domains, are boxed in light blue. Asterisks mark the conserved asparagine and tyrosine residues that have been mutated in this study in hsZBP1, as well as a conserved tryptophan. Residues that form the hydrophobic core are boxed in green. Residues that are neither DNA contacting nor structural but match the consensus sequence are highlighted in yellow. Isoleucine 335 in Zβ ADAR1 is highlighted in red. (B) The exon composition of the most prominent ZBP1 splice variants ZBP1full and ZBP1ΔZα as well as that of artificial constructs are shown. Exon 7 is rarely found in mRNA. Exon 9 contains an alternative termination site ( 22 ). ZBP1full and ZBP1ΔZα have been expressed as un-tagged or GFP tagged proteins in HeLa cells. ZBP1ΔZβ, ZBP1ΔZαΔZβ, ZBP1E1-5 and ZBP1E1-5ΔZα were expressed as GFP-tagged proteins. Schematic representation of the exon composition of constructs expressed from pET28a (p28) vectors in E.coli are shown in (C).
Figure Legend Snippet: Comparison of Zα domains ( A ) and schematic representation of constructs used for transfections ( B ) and protein expression ( C ). Comparison of Zα domains of human (hs) and mouse (mm) ZBP1, human ADAR1, zebrafish (dr) PKZ, vaccinia virus (vv) E3L and yaba-like disease virus (yldv) E3L is shown (A). The structures of the mouse (mm) Zα ZBP1 , human (hs) Zα ADAR1 , yaba-like disease virus (yldv) Zα E3L domains have been determined in complex with Z-DNA. Residues that make contact with Z-DNA, or the analogous residues in other Zα domains, are boxed in light blue. Asterisks mark the conserved asparagine and tyrosine residues that have been mutated in this study in hsZBP1, as well as a conserved tryptophan. Residues that form the hydrophobic core are boxed in green. Residues that are neither DNA contacting nor structural but match the consensus sequence are highlighted in yellow. Isoleucine 335 in Zβ ADAR1 is highlighted in red. (B) The exon composition of the most prominent ZBP1 splice variants ZBP1full and ZBP1ΔZα as well as that of artificial constructs are shown. Exon 7 is rarely found in mRNA. Exon 9 contains an alternative termination site ( 22 ). ZBP1full and ZBP1ΔZα have been expressed as un-tagged or GFP tagged proteins in HeLa cells. ZBP1ΔZβ, ZBP1ΔZαΔZβ, ZBP1E1-5 and ZBP1E1-5ΔZα were expressed as GFP-tagged proteins. Schematic representation of the exon composition of constructs expressed from pET28a (p28) vectors in E.coli are shown in (C).

Techniques Used: Construct, Transfection, Expressing, Sequencing

15) Product Images from "High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression"

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression

Journal: BMC Veterinary Research

doi: 10.1186/1746-6148-10-115

A schematic diagram of the constructions used in this study and the alignment results for the expressed PiCV capsid gene. (A) The full-length wild-type and codon-optimized PiCV capsid protein genes were cloned independently into three expression vectors pET28a, pGEX-4T-1 or pET32a. The PiCV capsid protein with the various different fusion tags, namely a six-histidine (6xHis), a Glutathione-S-transferase (GST) and a Thioredoxin-coupled six-histidine (Trx) at its N-terminus were expressed by T7 or Tac promoter-driving after IPTG induction. (B) The nucleotide sequences were compared between the wild-type (WT) and the codon-optimized (OPT) PiCV capsid protein genes. The asterisk (*) represents the fact that the aligned nucleotides are identical.
Figure Legend Snippet: A schematic diagram of the constructions used in this study and the alignment results for the expressed PiCV capsid gene. (A) The full-length wild-type and codon-optimized PiCV capsid protein genes were cloned independently into three expression vectors pET28a, pGEX-4T-1 or pET32a. The PiCV capsid protein with the various different fusion tags, namely a six-histidine (6xHis), a Glutathione-S-transferase (GST) and a Thioredoxin-coupled six-histidine (Trx) at its N-terminus were expressed by T7 or Tac promoter-driving after IPTG induction. (B) The nucleotide sequences were compared between the wild-type (WT) and the codon-optimized (OPT) PiCV capsid protein genes. The asterisk (*) represents the fact that the aligned nucleotides are identical.

Techniques Used: Clone Assay, Expressing

16) Product Images from "Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications"

Article Title: Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-12-27

Schematic diagram of the constructs used for TAT-Apoptin protein expression. ( A ) Schematic representation of the TAT-Apoptin protein fused with different affinity tags together with the expression vectors used in this study. The designations of the TAT-Apoptin protein and its expression vectors are indicated, ( a ), ( b ), ( c ) and ( d ). The constructs, ( a ) and ( b ), contain the full-length TAT-VP3 gene cloned into the vectors pET28a and pGEX-4 T-1; these were used for expression of TAT-Apoptin protein with either a six-histidine (6 × His) tag or a glutathione-s-transferase (GST) tag at the N-terminus, respectively. Constructs ( c ) and ( d ) containing the TAT-VP3 gene that was codon-optimized; this was derived from construct ( b ) by replacing rare codons without altering the amino acid sequence. The codon-optimized TAT-VP3gene, TAT-VP3 opt , was then cloned into pET28a and pGEX-4 T-1. ( B ) Sequence comparison between the TAT-VP3 gene and the TAT-VP3 opt gene. The nucleotide sequences were compared between the original TAT-VP3 gene (wild type TAT-VP3) and the sequence of codon-optimized TAT-VP3 gene (TAT-VP3 Opt ) over the whole coding region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.
Figure Legend Snippet: Schematic diagram of the constructs used for TAT-Apoptin protein expression. ( A ) Schematic representation of the TAT-Apoptin protein fused with different affinity tags together with the expression vectors used in this study. The designations of the TAT-Apoptin protein and its expression vectors are indicated, ( a ), ( b ), ( c ) and ( d ). The constructs, ( a ) and ( b ), contain the full-length TAT-VP3 gene cloned into the vectors pET28a and pGEX-4 T-1; these were used for expression of TAT-Apoptin protein with either a six-histidine (6 × His) tag or a glutathione-s-transferase (GST) tag at the N-terminus, respectively. Constructs ( c ) and ( d ) containing the TAT-VP3 gene that was codon-optimized; this was derived from construct ( b ) by replacing rare codons without altering the amino acid sequence. The codon-optimized TAT-VP3gene, TAT-VP3 opt , was then cloned into pET28a and pGEX-4 T-1. ( B ) Sequence comparison between the TAT-VP3 gene and the TAT-VP3 opt gene. The nucleotide sequences were compared between the original TAT-VP3 gene (wild type TAT-VP3) and the sequence of codon-optimized TAT-VP3 gene (TAT-VP3 Opt ) over the whole coding region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.

Techniques Used: Construct, Expressing, Clone Assay, Derivative Assay, Sequencing

17) Product Images from "Recombination and identification of human alpha B-crystallin"

Article Title: Recombination and identification of human alpha B-crystallin

Journal: International Journal of Ophthalmology

doi: 10.18240/ijo.2018.12.06

Gel electrophoresis following Ecor l and XhoI double enzymatic digestion A: Recombinant plasmid PMD19-T-αB-crystallin; B: Recombinant plasmid pET28a-αB-crystallin. Lane 1: Marker; Lane 2: Target gene fragment.
Figure Legend Snippet: Gel electrophoresis following Ecor l and XhoI double enzymatic digestion A: Recombinant plasmid PMD19-T-αB-crystallin; B: Recombinant plasmid pET28a-αB-crystallin. Lane 1: Marker; Lane 2: Target gene fragment.

Techniques Used: Nucleic Acid Electrophoresis, Recombinant, Plasmid Preparation, Marker

18) Product Images from "Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein"

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

Journal: Iranian Journal of Biotechnology

doi: 10.21859/ijb.1482

Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).
Figure Legend Snippet: Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).

Techniques Used: Construct, Nucleic Acid Electrophoresis, Plasmid Preparation, Marker

19) Product Images from "Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein"

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein

Journal: Iranian Journal of Biotechnology

doi: 10.21859/ijb.1482

Coomassie stained SDS-PAGE analysis of cell lysates from α-Luffin producing E. coli BL21 (DE3) clones in different temperatures, time and mode of culture. Panel A: LB batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates before IPTG induction; Lane 3, Cell lysates 4h post induction; Lane 4, Cell lysates 6h post induction; Lane 5, Cell lysates 8h post induction; Lane 6, Cell lysates 12h post induction; Lane 7, Cell lysates 24h post induction; Lane 8, Protein marker SM0671. Panel B: EB Fed-batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Protein marker SM0671; Lane 3, Cell lysates before IPTG induction; Lane 4, Cell lysates 4h post induction; Lane 5, Cell lysates 6h post induction; Lane 6, Cell lysates 8h post induction; Lane 7, Cell lysates total protein 10h post induction; Lane 8, Cell lysates total protein 12h post induction; Lane 9, Cell lysates total protein 24h post induction. Panel C: LB batch culture mode in 25°C. Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates total protein before IPTG induction; Lane 3, Cell lysates total protein 4h post induction; Lane 4, Cell lysates total protein 6h post induction; Lane 5, Cell lysates total protein 8h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker SM0671. Panel D: EB Fed-batch culture mode in 25 °C . Lane1, Cell lysates total protein before IPTG induction; Lane 2, Cell lysates total protein 4h post induction; Lane 3, Cell lysates total protein 6h post induction; Lane 4, Cell lysates total protein 8h post induction; Lane 5, Cell lysates total protein 10h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker Thermo science #26610. All samples were diluted to equal cell concentration before lysis and loading on gel. The position of 28.8kDa His-α-Luffin is indicated by arrows.
Figure Legend Snippet: Coomassie stained SDS-PAGE analysis of cell lysates from α-Luffin producing E. coli BL21 (DE3) clones in different temperatures, time and mode of culture. Panel A: LB batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates before IPTG induction; Lane 3, Cell lysates 4h post induction; Lane 4, Cell lysates 6h post induction; Lane 5, Cell lysates 8h post induction; Lane 6, Cell lysates 12h post induction; Lane 7, Cell lysates 24h post induction; Lane 8, Protein marker SM0671. Panel B: EB Fed-batch culture mode in 30 °C . Lane1, Control ( E. coli /pET28a only); Lane 2, Protein marker SM0671; Lane 3, Cell lysates before IPTG induction; Lane 4, Cell lysates 4h post induction; Lane 5, Cell lysates 6h post induction; Lane 6, Cell lysates 8h post induction; Lane 7, Cell lysates total protein 10h post induction; Lane 8, Cell lysates total protein 12h post induction; Lane 9, Cell lysates total protein 24h post induction. Panel C: LB batch culture mode in 25°C. Lane1, Control ( E. coli /pET28a only); Lane 2, Cell lysates total protein before IPTG induction; Lane 3, Cell lysates total protein 4h post induction; Lane 4, Cell lysates total protein 6h post induction; Lane 5, Cell lysates total protein 8h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker SM0671. Panel D: EB Fed-batch culture mode in 25 °C . Lane1, Cell lysates total protein before IPTG induction; Lane 2, Cell lysates total protein 4h post induction; Lane 3, Cell lysates total protein 6h post induction; Lane 4, Cell lysates total protein 8h post induction; Lane 5, Cell lysates total protein 10h post induction; Lane 6, Cell lysates total protein 12h post induction; Lane 7, Cell lysates total protein 24h post induction; Lane 8, Protein marker Thermo science #26610. All samples were diluted to equal cell concentration before lysis and loading on gel. The position of 28.8kDa His-α-Luffin is indicated by arrows.

Techniques Used: Staining, SDS Page, Marker, Concentration Assay, Lysis

Western blot analysis of total cell lysates and purified α-Luffin. A: Western blot analysis on total cell lysates of α-Luffin producing E. coli BL21 clones cultured in the fed-batch mode. Proteins were visualized with an anti-His antibody conjugated with alkaline phosphatase and DAB substrate. Lane 1, Control positive, an 18kDa His-tagged protein; Lane 2, protein marker Thermo science #26616; Lanes 3 and 4, E. coli BL21 cell lysates 6h after induction; Lane 5, Control negative before induction; Lane 6, Control negative E. coli /pET28a alone. B: SDS-PAGE (Lanes 1-8) and Western blot (Lanes 9-12) analysis of purified α-Luffin from fed batch process. Lane 1, Fed-batch soluble fraction (initial sample); Lane 2, NTA flow through sample; Lanes 3-7, Elution fractions from NTA column representing purified α-Luffin; Lane 9, His-tagged control protein (18 kDa); Lanes 11 and 12, NTA purified α-Luffin from fed-batch soluble fraction; Lanes 8 and 10, Protein size marker Thermo science #26610.
Figure Legend Snippet: Western blot analysis of total cell lysates and purified α-Luffin. A: Western blot analysis on total cell lysates of α-Luffin producing E. coli BL21 clones cultured in the fed-batch mode. Proteins were visualized with an anti-His antibody conjugated with alkaline phosphatase and DAB substrate. Lane 1, Control positive, an 18kDa His-tagged protein; Lane 2, protein marker Thermo science #26616; Lanes 3 and 4, E. coli BL21 cell lysates 6h after induction; Lane 5, Control negative before induction; Lane 6, Control negative E. coli /pET28a alone. B: SDS-PAGE (Lanes 1-8) and Western blot (Lanes 9-12) analysis of purified α-Luffin from fed batch process. Lane 1, Fed-batch soluble fraction (initial sample); Lane 2, NTA flow through sample; Lanes 3-7, Elution fractions from NTA column representing purified α-Luffin; Lane 9, His-tagged control protein (18 kDa); Lanes 11 and 12, NTA purified α-Luffin from fed-batch soluble fraction; Lanes 8 and 10, Protein size marker Thermo science #26610.

Techniques Used: Western Blot, Purification, Clone Assay, Cell Culture, Marker, SDS Page, Flow Cytometry

Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).
Figure Legend Snippet: Restriction analysis of pET28a-α-Luffin construct by gel electrophoresis. Lane 1 and 2, NdeI/XhoI digested plasmids (different clones); Lane 3, Undigested plasmid; Lane 4, DNA size marker (1-kb DNA ladder Fermentas® SM0311).

Techniques Used: Construct, Nucleic Acid Electrophoresis, Plasmid Preparation, Marker

20) Product Images from "GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance"

Article Title: GLYI and D-LDH play key role in methylglyoxal detoxification and abiotic stress tolerance

Journal: Scientific Reports

doi: 10.1038/s41598-018-23806-4

Heterologous expression of MG detoxification enzymes in E. coli provides varying tolerance to various abiotic stresses: The BL21 E. coli cells containing the constructs of MG detoxification genes (pET28a-AtGLYI, pET28a-AtGLYII and pET28a-AtD-LDH) were grown in presence of different abiotic stresses such as ( A ) salinity (200 mM NaCl), ( B ) oxidative (5 mM H 2 O 2 ) and ( C ) exogenous MG (0.5 mM MG) and their growth was monitored. Cells containing empty vector were used as control.
Figure Legend Snippet: Heterologous expression of MG detoxification enzymes in E. coli provides varying tolerance to various abiotic stresses: The BL21 E. coli cells containing the constructs of MG detoxification genes (pET28a-AtGLYI, pET28a-AtGLYII and pET28a-AtD-LDH) were grown in presence of different abiotic stresses such as ( A ) salinity (200 mM NaCl), ( B ) oxidative (5 mM H 2 O 2 ) and ( C ) exogenous MG (0.5 mM MG) and their growth was monitored. Cells containing empty vector were used as control.

Techniques Used: Expressing, Construct, Plasmid Preparation

Related Articles

Clone Assay:

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: Cloning of the recombinant HA1 protein The HA1 sequence, encompassing amino acids 1-320 of the A/Indonesia/05/05 strain of H5N1 influenza virus (AFM78567.1), was optimized for expression in E. coli by GENSCRIPT (www.Genscript.com) and synthesized by GENERAY. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector.

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: E. coli PLK831 [F-, gal-25 , λ− , ΔtrpE63 , pyrF287 , fnr-1 , IN(rrnD-rrnE)1 , rpsL195 (strR), iclR7 (Const), trpR72 (Am)] and RS2 [F-, gal-25 , λ− , topA10 , pyrF287 , fnr-1 , rpsL195 (strR), iclR7 (Const), trpR72 (Am)] were provided from the Coli Genetic Stock Center. .. The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The yjhQ gene was also amplified and cloned into pBAD24 ( ) and pCold-PST ( ).

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: Then 0.5 ml of the overnight culture were inoculated into 50 ml LB medium and grown at 37°C for around 3 h by which time the optical density of culture had reach 0.5 of OD600 for cell transformation or protein expression. .. A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: The resultant products were cloned into pTA2 vector (Toyobo) directly and sequenced to confirm PCR fidelity. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively. .. And the PCP gene was cloned into the pET28a-SUMO (Novagen), resulting in a PCP -containing-plasmid pYY0098.

Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase
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Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
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Amplification:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The amplicon with the desired band size was purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pGEMT (Promega, USA), as per the manufacturer’s instructions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
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Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. To generate the plasmid harboring the codon optimized nucleotide sequence, a codon optimized fragment encoding the first 107 amino acid residues of the N-terminus of the VP1 capsid protein was fused with the C-terminus of the VP1 protein using the 5' and 3' ends of above two DNA fragments and overlapping PCR [ ].

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
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Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: The purified amplicon was ligated into a pGEMT-Easy vector (Promega, USA) per manufacturer’s instructions, followed by transformation into competent E. coli DH5α. .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
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Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
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Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
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Synthesized:

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: NdeI and XhoI restriction sites were designed at 5′ and 3′ ends of the gene, respectively. .. After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: A poly-histidine affinity purification tag (6His) was also added to the C-terminus. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: Based on the Tβ 4 amino acid sequence, a Tβ 4 gene was designed and synthesized [ ], and DNA fragment of the Tβ 4 gene was subcloned into the pUC57 plasmid (termed pUC57-Tβ 4). .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: Moreover, we provide a plethora of data ranging from gene and protein expression, and substrate specificity to functional genomics, which we believe are an excellent basis for future research. .. Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. NUDT21 and NUDT22 cDNAs were purchased from Source BioScience and were subcloned into pET28a(+).

Construct:

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: Start codon (AUG) and stop codon (UAA) were added into the 5´ and 3´ ends of the construct, respectively and recognition sites of BamHI and XhoI restriction enzymes were introduced into the 5´ and 3´ ends of the synthetic gene, respectively. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector.

Article Title: Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens
Article Snippet: Forward and reverse primers for amplifying ispE of B. mallei , S. Typhi , and V. cholerae were designed to remove the five C-terminal amino acids , resulting in generation of three truncated versions of bacterial IspE enzymes. .. Expression constructs, pET28a(+)∷ mBME , pET28a(+)∷ mSTE , and pET28a(+)∷ mVCE were generated as described above. .. Recombinant Rv1011 I through VIII, mBME, mSTE, and mVCE were expressed and purified as previously described ( ; ).

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The topB , topA and truncated mutants, topA67 and topA14 were also amplified by PCR and cloned into pET28a to express the N-terminal His-tagged TopB, TopA, TopA67 (67 kDa; residues 1–597) and TopA14 (14 kDa; residues 745–865), respectively.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The PCR product was gel purified and ligated into the plasmid pMD18 (Takara Biotechnology, Dalian, China) and termed pMD18-6 × his-4×Tβ 4. .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China). .. The recombinant pET28a-6 × his-4 × Tβ 4 was transformed into E. coli strain BL21 by a thermal impulse at 42°C for 90 seconds.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen).

Electrophoresis:

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Incubation:

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The PCR product was gel purified and ligated into the plasmid pMD18 (Takara Biotechnology, Dalian, China) and termed pMD18-6 × his-4×Tβ 4. .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China). .. The recombinant pET28a-6 × his-4 × Tβ 4 was transformed into E. coli strain BL21 by a thermal impulse at 42°C for 90 seconds.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. To obtain Acas RegA protein, plasmid pET-AcRegA was transformed into E.coli BL21DE3.

Expressing:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: Paragraph title: Construction of recombinant burI expression plasmids ... A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: NdeI and XhoI restriction sites were designed at 5′ and 3′ ends of the gene, respectively. .. After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: A poly-histidine affinity purification tag (6His) was also added to the C-terminus. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

Article Title: Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens
Article Snippet: Forward and reverse primers for amplifying ispE of B. mallei , S. Typhi , and V. cholerae were designed to remove the five C-terminal amino acids , resulting in generation of three truncated versions of bacterial IspE enzymes. .. Expression constructs, pET28a(+)∷ mBME , pET28a(+)∷ mSTE , and pET28a(+)∷ mVCE were generated as described above. .. Recombinant Rv1011 I through VIII, mBME, mSTE, and mVCE were expressed and purified as previously described ( ; ).

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Paragraph title: Construction of Recombinant AciI Expression Plasmids ... The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Paragraph title: Construction of Recombinant cneI Expression Plasmids ... This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase
Article Snippet: P. putida ATCC12633 was used as the source of creatinase gene (Persian Type Culture Collection (IRO-ST PTCC, Iran)). .. E. coli DH5α strain (Novagen, Germany) and E. coli BL21 (DE3) were used as cloning and expression hosts and the pET28a(+) vector (Novagen, Germany) was used as expression vector. .. Kanamycin was obtained from duchefa, Taq polymerase, 2x PCR master mixes, 1 kb DNA Ladder, NheI and XhoI Restriction Enzymes, T4 DNA Ligase, IPTG (isopropyl-β-D-thiogalactopyranoside) and Protein ladder were obtained from fermentas.

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: Furthermore, an attempt was made to evaluate autophagy induction by chemo-selective ligation of CPA and CPB with α-alumina nanoparticles in peritoneal macrophages of BALB/c mice using cytotoxicity assay. .. The material in this study included GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia), Clone JET PCR Cloning Kit (Thermo Fisher Scientific, Inc., USA), pET28a, expression vector (Novagen, USA), IPTG (Sinaclon, Iran), kanamycin (Sigma, Germany), Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, USA), Ni–NTA agarose (Qiagen, USA), Anti-His antibody (Bioligand, USA), IgG-horseradish peroxidase (HPR)-conjugated secondary antibody (Abcam, USA), EDC (G-Biosciences, USA), SANH (Bioworld, USA), FITC (Sigma-Aldrich, USA), 3-MA (Sigma, Germany), and rapamycin (Sigma, Germany). .. In this research study, BALB/c mice were used and kept according to the Guide for the Use and Care of Laboratory Animals as approved by the Ethics Committee of Tarbiat Modares University.

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: The resulting E. coli culture was used to isolate the plasmid with QIAprep Spin Miniprep Kit (Qiagin, Hilden, Germany). .. A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. .. The forward primer was 5′-CG GAATTC ATGGGTTCACAAAGTGAAACCG-3′, in which the underlined region is an introduced EcoR I restriction site.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: Paragraph title: Cloning and expression of Acas RegA ... The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: Moreover, we provide a plethora of data ranging from gene and protein expression, and substrate specificity to functional genomics, which we believe are an excellent basis for future research. .. Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. NUDT21 and NUDT22 cDNAs were purchased from Source BioScience and were subcloned into pET28a(+).

Transformation Assay:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The resulting recombinant plasmid (designated pGEMT-burI ) was transformed into E. coli JM109 ( ). .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The resulting recombinant plasmid (designated pDrive-aciI) was transformed into E. coli DH5α ( ). .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: The purified amplicon was ligated into a pGEMT-Easy vector (Promega, USA) per manufacturer’s instructions, followed by transformation into competent E. coli DH5α. .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The BL21 cell strain harboring pET28a-6 × his-4 × Tβ 4 was cultured in a 15 mL flask with 3 mL LB broth liquid medium (pH = 7.5) containing kanamycin (100 μ g/mL) in a shaker (220 rpm) at 37°C for overnight. .. A similar culture of the BL21 cell strain transformed with pET28a was also grown and used as a negative control. .. Both cultures (200 μ L) were added to 50 mL fresh LB medium with kanamycin (100 μ g/mL) and cultured to the OD600 = 0.5 in a 37°C shaker (220 rpm).

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The expected hybridization signal at 30.87 kDa was detected at different induction times from 0 h to 8 h in BL 21 harboring pET28a-6 × his-4 × Tβ 4; however the signal was much weaker in 0 h sample. .. As expected, no immunoreactive band corresponding 4 × Tβ 4 was detected in the negative control BL21 samples form cultures transformed with pET28a ( ). .. In order to confirm the bioactivity of the recombinant 4 × Tβ 4, the E. coli -derived 4 × Tβ 4 protein was purified by the Ni-NTA resin and examined by SDS-PAGE gel.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The DNA sequence was determined from three clones and showed an open reading frame of 1863 bp.

Gel Purification:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The resulting recombinant plasmid (designated pGEMT-burI ) was transformed into E. coli JM109 ( ). .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The resulting recombinant plasmid (designated pDrive-aciI) was transformed into E. coli DH5α ( ). .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. This produced the recombinant plasmid designated pET28a-aciI.

Cell Culture:

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. They subsequently transformed into E. coli strain BL21 (DE3) for expression analysis.

Generated:

Article Title: Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens
Article Snippet: Forward and reverse primers for amplifying ispE of B. mallei , S. Typhi , and V. cholerae were designed to remove the five C-terminal amino acids , resulting in generation of three truncated versions of bacterial IspE enzymes. .. Expression constructs, pET28a(+)∷ mBME , pET28a(+)∷ mSTE , and pET28a(+)∷ mVCE were generated as described above. .. Recombinant Rv1011 I through VIII, mBME, mSTE, and mVCE were expressed and purified as previously described ( ; ).

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The pUC57-4 × Tβ 4 containing four repeats of the Tβ 4 gene was also generated based on the isocaudamer property of Spe I and Xba I. .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

DNA Sequencing:

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. This produced the recombinant plasmid designated pET28a-aciI.

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: Recombinant pJET1.2 plasmids were verified using common colony-Polymerase Chain Reaction (PCR) and restriction enzyme digestion followed by DNA sequencing. .. CPB- and CPA-coding DNA was then sub-cloned by insertion between BamHI and HindIII sites in pET28a (expression vector) (Novagen, USA).

Polymerase Chain Reaction:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR product was verified using agarose gel electrophoresis followed by ethidium bromide (Sigma, St. Louis, MO) staining. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: E. coli PLK831 [F-, gal-25 , λ− , ΔtrpE63 , pyrF287 , fnr-1 , IN(rrnD-rrnE)1 , rpsL195 (strR), iclR7 (Const), trpR72 (Am)] and RS2 [F-, gal-25 , λ− , topA10 , pyrF287 , fnr-1 , rpsL195 (strR), iclR7 (Const), trpR72 (Am)] were provided from the Coli Genetic Stock Center. .. The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The yjhQ gene was also amplified and cloned into pBAD24 ( ) and pCold-PST ( ).

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. This construct carries the VP1 gene flanked by the N-terminal glutathione-S-transferase (GST) tag and will express a GST-VP1 fusion protein.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: The resultant products were cloned into pTA2 vector (Toyobo) directly and sequenced to confirm PCR fidelity. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Upon running agarose gel electrophoresis, the desired amplicon was extracted using Wizard® SV Gel and PCR Clean-Up System (Promega, USA). .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: Recombinant pJET1.2 plasmids were verified using common colony-Polymerase Chain Reaction (PCR) and restriction enzyme digestion followed by DNA sequencing. .. CPB- and CPA-coding DNA was then sub-cloned by insertion between BamHI and HindIII sites in pET28a (expression vector) (Novagen, USA).

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The PCR product was gel purified and ligated into the plasmid pMD18 (Takara Biotechnology, Dalian, China) and termed pMD18-6 × his-4×Tβ 4. .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: Furthermore, an attempt was made to evaluate autophagy induction by chemo-selective ligation of CPA and CPB with α-alumina nanoparticles in peritoneal macrophages of BALB/c mice using cytotoxicity assay. .. The material in this study included GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia), Clone JET PCR Cloning Kit (Thermo Fisher Scientific, Inc., USA), pET28a, expression vector (Novagen, USA), IPTG (Sinaclon, Iran), kanamycin (Sigma, Germany), Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, USA), Ni–NTA agarose (Qiagen, USA), Anti-His antibody (Bioligand, USA), IgG-horseradish peroxidase (HPR)-conjugated secondary antibody (Abcam, USA), EDC (G-Biosciences, USA), SANH (Bioworld, USA), FITC (Sigma-Aldrich, USA), 3-MA (Sigma, Germany), and rapamycin (Sigma, Germany). .. In this research study, BALB/c mice were used and kept according to the Guide for the Use and Care of Laboratory Animals as approved by the Ethics Committee of Tarbiat Modares University.

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. .. A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector.

Affinity Purification:

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: A poly-histidine affinity purification tag (6His) was also added to the C-terminus. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector.

Recombinant:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The resulting recombinant plasmid (designated pGEMT-burI ) was transformed into E. coli JM109 ( ). .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: Cloning of the recombinant HA1 protein The HA1 sequence, encompassing amino acids 1-320 of the A/Indonesia/05/05 strain of H5N1 influenza virus (AFM78567.1), was optimized for expression in E. coli by GENSCRIPT (www.Genscript.com) and synthesized by GENERAY. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Paragraph title: Construction of Recombinant AciI Expression Plasmids ... The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Paragraph title: Construction of Recombinant cneI Expression Plasmids ... This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: Recombinant pJET1.2 plasmids were verified using common colony-Polymerase Chain Reaction (PCR) and restriction enzyme digestion followed by DNA sequencing. .. CPB- and CPA-coding DNA was then sub-cloned by insertion between BamHI and HindIII sites in pET28a (expression vector) (Novagen, USA).

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: To facilitate the purification of the recombinant 4 × Tβ 4 protein, a DNA sequence which is encoding six histidines was added at 5′ end of the 4 × Tβ 4 concatemer by PCR technique using the primers of Tβ 4F1 (5′-ggggatccatgcaccaccaccaccaccacggtaccatgtctagaatgtctga-3′) and Tβ 4R1 (5′-ccgagctcttaactagtcataga-3′). .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: Paragraph title: 4.2. Expression of Recombinant VbDFR in E. coli and Purification ... A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector.

Molecular Weight:

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: Bioinformatic analysis of HA1 antigen Physio-biochemical characteristics of HA1 antigen including isoelectric point, theoretical molecular weight, aliphatic index and GRAVY were analyzed through EXPASY (ProtParam tool). .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector.

DNA Extraction:

Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase
Article Snippet: E. coli DH5α strain (Novagen, Germany) and E. coli BL21 (DE3) were used as cloning and expression hosts and the pET28a(+) vector (Novagen, Germany) was used as expression vector. .. E. coli DH5α strain (Novagen, Germany) and E. coli BL21 (DE3) were used as cloning and expression hosts and the pET28a(+) vector (Novagen, Germany) was used as expression vector.

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: The CPA (650 bp ) and CPB (950 bp ) blunt-ended PCR products were gel purified using GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia) and cloned in pJET1.2 plasmid vector using CloneJET PCR Cloning Kit (K1231; Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s protocol. .. CPB- and CPA-coding DNA was then sub-cloned by insertion between BamHI and HindIII sites in pET28a (expression vector) (Novagen, USA).

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: Furthermore, an attempt was made to evaluate autophagy induction by chemo-selective ligation of CPA and CPB with α-alumina nanoparticles in peritoneal macrophages of BALB/c mice using cytotoxicity assay. .. The material in this study included GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia), Clone JET PCR Cloning Kit (Thermo Fisher Scientific, Inc., USA), pET28a, expression vector (Novagen, USA), IPTG (Sinaclon, Iran), kanamycin (Sigma, Germany), Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, USA), Ni–NTA agarose (Qiagen, USA), Anti-His antibody (Bioligand, USA), IgG-horseradish peroxidase (HPR)-conjugated secondary antibody (Abcam, USA), EDC (G-Biosciences, USA), SANH (Bioworld, USA), FITC (Sigma-Aldrich, USA), 3-MA (Sigma, Germany), and rapamycin (Sigma, Germany). .. In this research study, BALB/c mice were used and kept according to the Guide for the Use and Care of Laboratory Animals as approved by the Ethics Committee of Tarbiat Modares University.

Isolation:

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: Total Acas RNA was isolated using the Qiagen RNeasy Mini Kit and reverse transcribed with SuperScript III First-Strand Synthesis System (Invitrogen, Paisley, UK), using primers AcRegAF and AcRegAR, that contained NheI and EcoRI sites respectively, followed by cDNA amplification with Phusion High-Fidelity DNA Polymerase (NEB, Ipswich, MA). .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: Moreover, we provide a plethora of data ranging from gene and protein expression, and substrate specificity to functional genomics, which we believe are an excellent basis for future research. .. Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. NUDT21 and NUDT22 cDNAs were purchased from Source BioScience and were subcloned into pET28a(+).

Subcloning:

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: Paragraph title: 3.2. Subcloning of α-Luffin Gene Fragment ... After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag.

Size-exclusion Chromatography:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR cycles consist of an initial denaturation at 95 °C for 5 min, followed by 30 cycles at 95 °C for 30 s, annealing at 55 °C for 40 s and extension at 72 °C for 40 sec, and a final extension at 72 °C for 5 min. Sterile deionised water was used as the negative control in all PCR reactions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Purification:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The amplicon with the desired band size was purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pGEMT (Promega, USA), as per the manufacturer’s instructions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: Paragraph title: Production and purification of ACPs/PCP ... Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: The recombinant vector, designated pGEMT-Easy_cneI , was purified and linearized with both NcoI and BamHI enzymes (NEB, USA). .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes. .. The resulting recombinant plasmid was designated pET28a_cneI .

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: The CPA (650 bp ) and CPB (950 bp ) blunt-ended PCR products were gel purified using GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia) and cloned in pJET1.2 plasmid vector using CloneJET PCR Cloning Kit (K1231; Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s protocol. .. CPB- and CPA-coding DNA was then sub-cloned by insertion between BamHI and HindIII sites in pET28a (expression vector) (Novagen, USA).

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The PCR product was gel purified and ligated into the plasmid pMD18 (Takara Biotechnology, Dalian, China) and termed pMD18-6 × his-4×Tβ 4. .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: Paragraph title: 4.2. Expression of Recombinant VbDFR in E. coli and Purification ... A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The culture was then diluted 1:40 in LB, incubated for 2 h at 30 °C and supplemented with 1 mM IPTG.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. Expression constructs of NUDT3 (aa 8–172), NUDT5, the catalytic subunit of NUDT6, NUDT10 (variant AAH50700), NUDT11 (aa 13–164), NUDT12, NUDT14, NUDT15, and NUDT16 (variant AAH31215) in pNIC28 were kind gifts from SGC Stockholm.

Sequencing:

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: A poly-histidine affinity purification tag (6His) was also added to the C-terminus. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ]. .. This construct carries the VP1 gene flanked by the N-terminal glutathione-S-transferase (GST) tag and will express a GST-VP1 fusion protein.

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. This produced the recombinant plasmid designated pET28a-aciI.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes. .. The resulting recombinant plasmid was designated pET28a_cneI .

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: To facilitate the purification of the recombinant 4 × Tβ 4 protein, a DNA sequence which is encoding six histidines was added at 5′ end of the 4 × Tβ 4 concatemer by PCR technique using the primers of Tβ 4F1 (5′-ggggatccatgcaccaccaccaccaccacggtaccatgtctagaatgtctga-3′) and Tβ 4R1 (5′-ccgagctcttaactagtcataga-3′). .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: 2.1.2 The partially assembled Acas genome http://blast.hgsc.bcm.tmc.edu/blast.hgsc?organism=AcastellaniNeff was queried by tBlastn with Ddis RegA, yielding hits on 3 contigs, which after assembly yielded about 3.3 kb of coding sequence homologous to the query sequence, but containing many introns. .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag.

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen).

Protein Extraction:

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The culture was then diluted 1:40 in LB, incubated for 2 h at 30 °C and supplemented with 1 mM IPTG.

Staining:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR product was verified using agarose gel electrophoresis followed by ethidium bromide (Sigma, St. Louis, MO) staining. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Plasmid Preparation:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The resulting recombinant plasmid (designated pGEMT-burI ) was transformed into E. coli JM109 ( ). .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI . .. Verification for the correct insert cloned into pGEMT and pET28a plasmids was done by automated Sanger DNA sequencing.

Article Title: Optimization of EnBase Fed-Batch Cultivation to Improve Soluble Fraction Ratio of α-Luffin Ribosome Inactivating Protein
Article Snippet: NdeI and XhoI restriction sites were designed at 5′ and 3′ ends of the gene, respectively. .. After restriction digestion analysis, the confirmed synthesized gene was subcloned into the expression vector, pET28a (Novagen, USA) under the control of T7 promoter and fused to the 6x-histidine tag. .. Recombinant clone was selected through further restriction digestions and sequencing with T7 sequencing forward and reverse primers.

Article Title: Expression of HA1 antigen of H5N1 influenza virus as a potent candidate for vaccine in bacterial system
Article Snippet: A poly-histidine affinity purification tag (6His) was also added to the C-terminus. .. The synthesized sequence was removed from the pUC57 vector by digestion with BamHI and XhoI and then inserted into the linearized pET28a (+) expression vector (Novagen, USA) using T4 DNA ligase (Fermentas, USA), yielding pET28a-HA1 vector. .. The resulting vector transformed into E. coli strain DH5α cells through heat shock transformation method and they were selected on LB agar plate containing 50 µg.m-1 ampicillin.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
Article Snippet: Paragraph title: Plasmid construction ... A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure , panel a) (Lee et al., 2009) [ ].

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The resulting recombinant plasmid (designated pDrive-aciI) was transformed into E. coli DH5α ( ). .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. This produced the recombinant plasmid designated pET28a-aciI.

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: The resultant products were cloned into pTA2 vector (Toyobo) directly and sequenced to confirm PCR fidelity. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: The recombinant vector, designated pGEMT-Easy_cneI , was purified and linearized with both NcoI and BamHI enzymes (NEB, USA). .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase
Article Snippet: P. putida ATCC12633 was used as the source of creatinase gene (Persian Type Culture Collection (IRO-ST PTCC, Iran)). .. E. coli DH5α strain (Novagen, Germany) and E. coli BL21 (DE3) were used as cloning and expression hosts and the pET28a(+) vector (Novagen, Germany) was used as expression vector. .. Kanamycin was obtained from duchefa, Taq polymerase, 2x PCR master mixes, 1 kb DNA Ladder, NheI and XhoI Restriction Enzymes, T4 DNA Ligase, IPTG (isopropyl-β-D-thiogalactopyranoside) and Protein ladder were obtained from fermentas.

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The PCR product was gel purified and ligated into the plasmid pMD18 (Takara Biotechnology, Dalian, China) and termed pMD18-6 × his-4×Tβ 4. .. Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China). .. The recombinant pET28a-6 × his-4 × Tβ 4 was transformed into E. coli strain BL21 by a thermal impulse at 42°C for 90 seconds.

Article Title: Alpha Alumina Nanoparticle Conjugation to Cysteine Peptidase A and B: An Efficient Method for Autophagy Induction
Article Snippet: Furthermore, an attempt was made to evaluate autophagy induction by chemo-selective ligation of CPA and CPB with α-alumina nanoparticles in peritoneal macrophages of BALB/c mice using cytotoxicity assay. .. The material in this study included GF-1 plasmid DNA extraction kit (Vivantis CO., Malaysia), Clone JET PCR Cloning Kit (Thermo Fisher Scientific, Inc., USA), pET28a, expression vector (Novagen, USA), IPTG (Sinaclon, Iran), kanamycin (Sigma, Germany), Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, USA), Ni–NTA agarose (Qiagen, USA), Anti-His antibody (Bioligand, USA), IgG-horseradish peroxidase (HPR)-conjugated secondary antibody (Abcam, USA), EDC (G-Biosciences, USA), SANH (Bioworld, USA), FITC (Sigma-Aldrich, USA), 3-MA (Sigma, Germany), and rapamycin (Sigma, Germany). .. In this research study, BALB/c mice were used and kept according to the Guide for the Use and Care of Laboratory Animals as approved by the Ethics Committee of Tarbiat Modares University.

Article Title: Molecular Cloning and Functional Characterization of a Dihydroflavonol 4-Reductase from Vitis bellula
Article Snippet: The resulting E. coli culture was used to isolate the plasmid with QIAprep Spin Miniprep Kit (Qiagin, Hilden, Germany). .. A pair of primers was designed to clone VbDFR to pET28a (+) (Novagen, Madison, WI, USA), a protein expression vector. .. The forward primer was 5′-CG GAATTC ATGGGTTCACAAAGTGAAACCG-3′, in which the underlined region is an introduced EcoR I restriction site.

Negative Control:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR cycles consist of an initial denaturation at 95 °C for 5 min, followed by 30 cycles at 95 °C for 30 s, annealing at 55 °C for 40 s and extension at 72 °C for 40 sec, and a final extension at 72 °C for 5 min. Sterile deionised water was used as the negative control in all PCR reactions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The BL21 cell strain harboring pET28a-6 × his-4 × Tβ 4 was cultured in a 15 mL flask with 3 mL LB broth liquid medium (pH = 7.5) containing kanamycin (100 μ g/mL) in a shaker (220 rpm) at 37°C for overnight. .. A similar culture of the BL21 cell strain transformed with pET28a was also grown and used as a negative control. .. Both cultures (200 μ L) were added to 50 mL fresh LB medium with kanamycin (100 μ g/mL) and cultured to the OD600 = 0.5 in a 37°C shaker (220 rpm).

Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice
Article Snippet: The expected hybridization signal at 30.87 kDa was detected at different induction times from 0 h to 8 h in BL 21 harboring pET28a-6 × his-4 × Tβ 4; however the signal was much weaker in 0 h sample. .. As expected, no immunoreactive band corresponding 4 × Tβ 4 was detected in the negative control BL21 samples form cultures transformed with pET28a ( ). .. In order to confirm the bioactivity of the recombinant 4 × Tβ 4, the E. coli -derived 4 × Tβ 4 protein was purified by the Ni-NTA resin and examined by SDS-PAGE gel.

Positron Emission Tomography:

Article Title: Characterization of Discrete Phosphopantetheinyl Transferases in Streptomyces tsukubaensis L19 Unveils a Complicate Phosphopantetheinylation Network
Article Snippet: The resultant products were cloned into pTA2 vector (Toyobo) directly and sequenced to confirm PCR fidelity. .. Then these genes were digested with Nde I/Hin dIII and cloned into the same sites of pET28a (Novagen), yielding three ACP -containing-plasmids pET-AACP and pYY0081-pYY0082, respectively. .. And the PCP gene was cloned into the pET28a-SUMO (Novagen), resulting in a PCP -containing-plasmid pYY0098.

Article Title: The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas
Article Snippet: Total Acas RNA was isolated using the Qiagen RNeasy Mini Kit and reverse transcribed with SuperScript III First-Strand Synthesis System (Invitrogen, Paisley, UK), using primers AcRegAF and AcRegAR, that contained NheI and EcoRI sites respectively, followed by cDNA amplification with Phusion High-Fidelity DNA Polymerase (NEB, Ipswich, MA). .. The cDNA was cloned after NheI/EcoRI digestion into similarly digested pET28a (Novagen, Leuven, Belgium), yielding plasmid pET-AcRegA, in which Acas RegA is fused at the N-terminus to a hexahis-tag. .. The DNA sequence was determined from three clones and showed an open reading frame of 1863 bp.

Agarose Gel Electrophoresis:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The PCR product was verified using agarose gel electrophoresis followed by ethidium bromide (Sigma, St. Louis, MO) staining. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Cloning and Characterization of the Autoinducer Synthase Gene from Lipid-Degrading Bacterium Cedecea neteri
Article Snippet: Upon running agarose gel electrophoresis, the desired amplicon was extracted using Wizard® SV Gel and PCR Clean-Up System (Promega, USA). .. This insert was then purified and ligated into pET28a (Novagen, Germany) that was linearized with the same restriction enzymes.

E. coli Genomic Assay:

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: E. coli PLK831 [F-, gal-25 , λ− , ΔtrpE63 , pyrF287 , fnr-1 , IN(rrnD-rrnE)1 , rpsL195 (strR), iclR7 (Const), trpR72 (Am)] and RS2 [F-, gal-25 , λ− , topA10 , pyrF287 , fnr-1 , rpsL195 (strR), iclR7 (Const), trpR72 (Am)] were provided from the Coli Genetic Stock Center. .. The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The yjhQ gene was also amplified and cloned into pBAD24 ( ) and pCold-PST ( ).

Strep-tag:

Article Title: An endogenous protein inhibitor, YjhX (TopAI), for topoisomerase I from Escherichia coli
Article Snippet: The topAI gene was amplified by polymerase chain reaction (PCR) using the E. coli genomic DNA as template and cloned into pET28a (Novagen). .. The topB , topA and truncated mutants, topA67 and topA14 were also amplified by PCR and cloned into pET28a to express the N-terminal His-tagged TopB, TopA, TopA67 (67 kDa; residues 1–597) and TopA14 (14 kDa; residues 745–865), respectively.

Gel Extraction:

Article Title: Whole genome sequencing enables the characterization of BurI, a LuxI homologue of Burkholderia cepacia strain GG4
Article Snippet: The amplicon with the desired band size was purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pGEMT (Promega, USA), as per the manufacturer’s instructions. .. A DNA fragment was excised from this recombinant plasmid by digestion with NcoI and XhoI followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes, to produce pET28a-burI .

Article Title: Unravelling the genome of long chain N-acylhomoserine lactone-producing Acinetobacter sp. strain GG2 and identification of its quorum sensing synthase gene
Article Snippet: Then, the PCR product was gel purified using QIAquick Gel Extraction kit (Qiagen, Germany) and ligated to pDrive cloning vector (Qiagen, Germany), according to the manufacturer’s instructions. .. The aciI gene was excised from this plasmid by digestion with NcoI and XhoI (Promega, USA) followed by gel purification, and ligated into pET28a (Novagen, Germany) digested with the same enzymes.

Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase
Article Snippet: E. coli DH5α strain (Novagen, Germany) and E. coli BL21 (DE3) were used as cloning and expression hosts and the pET28a(+) vector (Novagen, Germany) was used as expression vector. .. E. coli DH5α strain (Novagen, Germany) and E. coli BL21 (DE3) were used as cloning and expression hosts and the pET28a(+) vector (Novagen, Germany) was used as expression vector.

Variant Assay:

Article Title: A comprehensive structural, biochemical and biological profiling of the human NUDIX hydrolase family
Article Snippet: Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen). .. Complementary DNA encoding NUDT4 (DIPP2α) and NUDT9 was amplified from cDNA synthesized from RNA isolated in house from HL60 cells and subcloned into pET28a(+) (Novagen). cDNAs encoding NUDT1 (MTH1), NUDT2, NUDT7, NUDT17, and NUDT18 were codon optimized for E. coli expression and purchased from GeneArt (Life Technologies) and subcloned into pET28a(+) (Novagen).

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    Millipore pet28a
    Diagramme of <t>pET28a-6</t> × his-4 × T β 4. lac I, lac repressor coding gene; Kan: kanamycin coding gene; 6 × his, a DNA sequence encoding six histidines; 4 × Tβ 4, four copies of a DNA sequence which encode Tβ 4 arranged in tandem; origin, DNA sequence of pBR322 origin of replication; f1 origin, DNA sequence of phage f1 origin of replication.
    Pet28a, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 804 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diagramme of pET28a-6 × his-4 × T β 4. lac I, lac repressor coding gene; Kan: kanamycin coding gene; 6 × his, a DNA sequence encoding six histidines; 4 × Tβ 4, four copies of a DNA sequence which encode Tβ 4 arranged in tandem; origin, DNA sequence of pBR322 origin of replication; f1 origin, DNA sequence of phage f1 origin of replication.

    Journal: BioMed Research International

    Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

    doi: 10.1155/2013/241721

    Figure Lengend Snippet: Diagramme of pET28a-6 × his-4 × T β 4. lac I, lac repressor coding gene; Kan: kanamycin coding gene; 6 × his, a DNA sequence encoding six histidines; 4 × Tβ 4, four copies of a DNA sequence which encode Tβ 4 arranged in tandem; origin, DNA sequence of pBR322 origin of replication; f1 origin, DNA sequence of phage f1 origin of replication.

    Article Snippet: Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

    Techniques: Sequencing

    (a) Expression of recombinant 4 × T β 4 in BL21 cells at different induction times. Lane M, middle-molecular mass protein markers; lanes from 1 to 5: induced expression of the recombinant 4 × T β 4 by IPTG (1 mM) at 0, 2, 4, 6, and 8 hs, respectively; lane 6: induced BL21 containing pET28a by IPTG (1 mM) for 6 hs. Red arrows indicate specific band of E. coli -derived 6 × his-×T β 4. (b) Western blot analysis of the E. coli -derived 6 × his-4 × T β 4. Lane M: middle-molecular-mass protein markers; lanes from 1 to 5: BL21 harboring pET28a-6 × his-4 × T β 4 after IPTG (1 mM) induction at 37°C for 0, 2, 4, 6, and 8 h, respectively; lane 6: induction of expression of the BL21 containing pET28a by IPTG (1 mM) at 37°C for 6 h. Red arrows indicate the specific hybridization signal for the E. coli -derived 6 × his-×T β 4.

    Journal: BioMed Research International

    Article Title: The Escherichia coli-Derived Thymosin β4 Concatemer Promotes Cell Proliferation and Healing Wound in Mice

    doi: 10.1155/2013/241721

    Figure Lengend Snippet: (a) Expression of recombinant 4 × T β 4 in BL21 cells at different induction times. Lane M, middle-molecular mass protein markers; lanes from 1 to 5: induced expression of the recombinant 4 × T β 4 by IPTG (1 mM) at 0, 2, 4, 6, and 8 hs, respectively; lane 6: induced BL21 containing pET28a by IPTG (1 mM) for 6 hs. Red arrows indicate specific band of E. coli -derived 6 × his-×T β 4. (b) Western blot analysis of the E. coli -derived 6 × his-4 × T β 4. Lane M: middle-molecular-mass protein markers; lanes from 1 to 5: BL21 harboring pET28a-6 × his-4 × T β 4 after IPTG (1 mM) induction at 37°C for 0, 2, 4, 6, and 8 h, respectively; lane 6: induction of expression of the BL21 containing pET28a by IPTG (1 mM) at 37°C for 6 h. Red arrows indicate the specific hybridization signal for the E. coli -derived 6 × his-×T β 4.

    Article Snippet: Subsequently, the plasmids pMD18-6 × his-4 × Tβ 4 and pET28a (Merck Millipore, Germany) were digested with Bam H I/Sac I, and the vector pET28a-6 × his-4 × Tβ 4 was constructed following incubation with T4 DNA ligase (Takara Biotechnology, Dalian, China).

    Techniques: Expressing, Recombinant, Derivative Assay, Western Blot, Hybridization