melittin gene  (New England Biolabs)


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    Standard Taq Mg free Reaction Buffer Pack
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    Standard Taq Mg free Reaction Buffer Pack 6 0 ml
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    New England Biolabs melittin gene
    Standard Taq Mg free Reaction Buffer Pack
    Standard Taq Mg free Reaction Buffer Pack 6 0 ml
    https://www.bioz.com/result/melittin gene/product/New England Biolabs
    Average 88 stars, based on 55 article reviews
    Price from $9.99 to $1999.99
    melittin gene - by Bioz Stars, 2021-02
    88/100 stars

    Images

    1) Product Images from "Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites"

    Article Title: Genetically Engineered Yeast Expressing a Lytic Peptide from Bee Venom (Melittin) Kills Symbiotic Protozoa in the Gut of Formosan Subterranean Termites

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0151675

    Test for gut integrity using workers fed with blue food dye on bait containing cellulose only (Control) or yeast expressing Melittin (Treatment).
    Figure Legend Snippet: Test for gut integrity using workers fed with blue food dye on bait containing cellulose only (Control) or yeast expressing Melittin (Treatment).

    Techniques Used: Expressing

    Changes in health of gut and protozoa of Formosan subterranean termite workers after feeding bait containing live yeast expressing Melittin, fluorescent protein (mPlum) or plain cellulose without yeast. Error bars represent 95% confidence intervals; non-overlapping confidence intervals signify significant difference among treatments.
    Figure Legend Snippet: Changes in health of gut and protozoa of Formosan subterranean termite workers after feeding bait containing live yeast expressing Melittin, fluorescent protein (mPlum) or plain cellulose without yeast. Error bars represent 95% confidence intervals; non-overlapping confidence intervals signify significant difference among treatments.

    Techniques Used: Expressing

    2) Product Images from "Glycogen Synthase Kinase 3 Phosphorylates RBL2/p130 during Quiescence"

    Article Title: Glycogen Synthase Kinase 3 Phosphorylates RBL2/p130 during Quiescence

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.20.8970-8980.2004

    Unique region of p130 contains three potential GSK3 phosphorylation sites. (A) Schematic structure of p130. The regions forming a pocket domain that is highly conserved among retinoblastoma family proteins are shown darkly shaded. The Loop region in the B-box of p130 is absent in pRb and has no homology with the corresponding region of p107. Residues matching the GSK3 phosphorylation consensus sequence are underlined. Positions of the sites (numbered from 1 to 6 for convenience) correspond to human p130. (B) GSK3 phosphorylates the Loop of p130 in vitro and requires priming phosphorylation. The GST-tagged S935-E1000 fragment (Loop) of p130 was absorbed on glutathione Sepharose beads and subjected to GSK3B (Gsk-3β) phosphorylation in the presence of [γ- 33 P]ATP either directly (lane 3) or after priming phosphorylation with purified MAPK and nonradioactive ATP followed by extensive washing of the beads (lane 4). A control reaction with MAPK1-prephosphorylated GST-Loop but without GSK3B shows that phosphorylation is mediated by GSK3B and not by residual MAPK1 activity (lane 2). Lane 1 demonstrates phosphorylation of GST-Loop by MAPK1 in the presence of [γ- 33 P]ATP. An autoradiogram shows phosphorylation of GST-Loop and autophosphorylation of GSK3B. (C) GSK3 phosphorylates p130 but not the 1,3,5/A or 1-6/A p130 mutants. HA-tagged p130 and the mutants were expressed in U-2 OS cells, immunoprecipitated, and incubated with purified GSK3B in the presence of [γ- 33 P]ATP. An identically prepared sample from vector-transfected cells was used as a control (Mock, lane 1). Reaction prod-ucts were resolved by SDS-PAGE (10% polyacrylamide gel), transferred to nitrocellulose, and subsequently analyzed by autoradiography and Western blot. The top and bottom panels show the autoradiograms of p130 phosphorylation and GSK3B autophosphorylation, respectively. The middle panel shows a Western blot with anti-HA antibody (WB: HA) confirming that comparable amounts of p130 and the mutants were present in each reaction. (D) GSK3 phosphorylates each of the three pairs of phosphorylation sites in the Loop region of p130. HA-tagged wild-type p130, 2,4/A, 2,6/A, and 4,6/A double mutants and the 2,4,6/A triple mutant were expressed and subjected to GSK3B phosphorylation as described for panel C. For each of the samples, a control reaction without GSK3B indicates that the phosphorylation is mediated by GSK3B and not by other p130-associated kinases (odd lanes). Reaction products were resolved by SDS-PAGE (10% polyacrylamide gel) and transferred to nitrocellulose. The top and bottom panels show the autoradiograms of p130 phosphorylation and GSK3B autophosphorylation, respectively. The middle panel shows a Western blot with anti-HA antibody confirming comparable amounts of p130 and the mutants were present in each reaction.
    Figure Legend Snippet: Unique region of p130 contains three potential GSK3 phosphorylation sites. (A) Schematic structure of p130. The regions forming a pocket domain that is highly conserved among retinoblastoma family proteins are shown darkly shaded. The Loop region in the B-box of p130 is absent in pRb and has no homology with the corresponding region of p107. Residues matching the GSK3 phosphorylation consensus sequence are underlined. Positions of the sites (numbered from 1 to 6 for convenience) correspond to human p130. (B) GSK3 phosphorylates the Loop of p130 in vitro and requires priming phosphorylation. The GST-tagged S935-E1000 fragment (Loop) of p130 was absorbed on glutathione Sepharose beads and subjected to GSK3B (Gsk-3β) phosphorylation in the presence of [γ- 33 P]ATP either directly (lane 3) or after priming phosphorylation with purified MAPK and nonradioactive ATP followed by extensive washing of the beads (lane 4). A control reaction with MAPK1-prephosphorylated GST-Loop but without GSK3B shows that phosphorylation is mediated by GSK3B and not by residual MAPK1 activity (lane 2). Lane 1 demonstrates phosphorylation of GST-Loop by MAPK1 in the presence of [γ- 33 P]ATP. An autoradiogram shows phosphorylation of GST-Loop and autophosphorylation of GSK3B. (C) GSK3 phosphorylates p130 but not the 1,3,5/A or 1-6/A p130 mutants. HA-tagged p130 and the mutants were expressed in U-2 OS cells, immunoprecipitated, and incubated with purified GSK3B in the presence of [γ- 33 P]ATP. An identically prepared sample from vector-transfected cells was used as a control (Mock, lane 1). Reaction prod-ucts were resolved by SDS-PAGE (10% polyacrylamide gel), transferred to nitrocellulose, and subsequently analyzed by autoradiography and Western blot. The top and bottom panels show the autoradiograms of p130 phosphorylation and GSK3B autophosphorylation, respectively. The middle panel shows a Western blot with anti-HA antibody (WB: HA) confirming that comparable amounts of p130 and the mutants were present in each reaction. (D) GSK3 phosphorylates each of the three pairs of phosphorylation sites in the Loop region of p130. HA-tagged wild-type p130, 2,4/A, 2,6/A, and 4,6/A double mutants and the 2,4,6/A triple mutant were expressed and subjected to GSK3B phosphorylation as described for panel C. For each of the samples, a control reaction without GSK3B indicates that the phosphorylation is mediated by GSK3B and not by other p130-associated kinases (odd lanes). Reaction products were resolved by SDS-PAGE (10% polyacrylamide gel) and transferred to nitrocellulose. The top and bottom panels show the autoradiograms of p130 phosphorylation and GSK3B autophosphorylation, respectively. The middle panel shows a Western blot with anti-HA antibody confirming comparable amounts of p130 and the mutants were present in each reaction.

    Techniques Used: Sequencing, In Vitro, Purification, Activity Assay, Immunoprecipitation, Incubation, Plasmid Preparation, Transfection, SDS Page, Autoradiography, Western Blot, Mutagenesis

    3) Product Images from "Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin"

    Article Title: Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.03151-13

    Cirsin PSI fused to the α-MF leader sequence is efficiently integrated into the K. lactis genome and secreted into culture medium. (A) Schematic representation of the α-MF_PSI(His) 6 (top) and α-MF_PSI(N86S)(His) 6 (bottom) fusion proteins. Both sequences comprise the α-MF preprodomain sequence and a hexahistidine tag. The two putative N-glycosylation sites in the α-MF leader sequence (NGT and NTT) are indicated. A putative N-glycosylation site (NET) is present in the α-MF_PSI(His) 6 sequence, while in the construct α-MF_PSI(N86S)(His) 6 , this glycosylation site is mutated (N86S). (B) Confirmation by PCR of integration of the expression cassettes into the K. lactis genome. The numbers correspond to the selected clones and “c” to the amplification product using genomic DNA from untransformed K. lactis strain GG799 (negative control). (Top) α-MF_PSI(His) 6 . (Bottom) α-MF_PSI(N86S)(His) 6 . (C) Western blot analysis of recombinant cirsin PSI expression/secretion into culture media with an anti-His antibody. The numbers above the gels correspond to selected clones shown in panel B, and the lane marked with a c corresponds to the analysis of the culture medium of untransformed K. lactis GG799 cells (negative control). (Left) α-MF_PSI(His) 6 transformants. (Right) α-MF_PSI(N86S)(His) 6 transformants.
    Figure Legend Snippet: Cirsin PSI fused to the α-MF leader sequence is efficiently integrated into the K. lactis genome and secreted into culture medium. (A) Schematic representation of the α-MF_PSI(His) 6 (top) and α-MF_PSI(N86S)(His) 6 (bottom) fusion proteins. Both sequences comprise the α-MF preprodomain sequence and a hexahistidine tag. The two putative N-glycosylation sites in the α-MF leader sequence (NGT and NTT) are indicated. A putative N-glycosylation site (NET) is present in the α-MF_PSI(His) 6 sequence, while in the construct α-MF_PSI(N86S)(His) 6 , this glycosylation site is mutated (N86S). (B) Confirmation by PCR of integration of the expression cassettes into the K. lactis genome. The numbers correspond to the selected clones and “c” to the amplification product using genomic DNA from untransformed K. lactis strain GG799 (negative control). (Top) α-MF_PSI(His) 6 . (Bottom) α-MF_PSI(N86S)(His) 6 . (C) Western blot analysis of recombinant cirsin PSI expression/secretion into culture media with an anti-His antibody. The numbers above the gels correspond to selected clones shown in panel B, and the lane marked with a c corresponds to the analysis of the culture medium of untransformed K. lactis GG799 cells (negative control). (Left) α-MF_PSI(His) 6 transformants. (Right) α-MF_PSI(N86S)(His) 6 transformants.

    Techniques Used: Sequencing, Construct, Polymerase Chain Reaction, Expressing, Clone Assay, Amplification, Negative Control, Western Blot, Recombinant

    Recombinant cirsin PSI displays antifungal activity. (A) Confrontation assays between the selected K. lactis transformant secreting wt PSI [SP_PSI(His) 6 ] and three phytopathogenic fungi ( A. alternata , L. infectoria , and D. biseptata ). Untransformed K. lactis (strain GG799) and a K. lactis transformant expressing GFP were used as negative controls. The antifungal activity is given by the percentage of mycelial growth inhibition {MGI = [(free growth of the fungi − growth of the fungi in the presence of yeast)/(free growth of the fungi)] × 100}. All assays were performed in duplicate. (B) Representative plates showing antifungal activity toward A. alternata and L. infectoria . (C and D) Disk diffusion test. Impregnated disks with a purified sample of recombinant nonglycosylated cirsin PSI (0.47 mg/ml) were assayed against the fungus A. alternata by evaluating the appearance of a clear zone of inhibition. The assay was performed in triplicate at two different pH values (pH 5.0 and 6.8) using the antifungal amphotericin B (0.25 mg/ml) as a positive control. (C) Antifungal activity is represented by the percentage of mycelial growth inhibition {MGI = [(growth of the fungi in the presence of sample buffer − growth of the fungi in the presence of PSI)/(growth of fungi in the presence of sample buffer)] × 100}. (D) Representative plates showing antifungal activity of PSI toward A. alternata . The error bars indicate standard deviations.
    Figure Legend Snippet: Recombinant cirsin PSI displays antifungal activity. (A) Confrontation assays between the selected K. lactis transformant secreting wt PSI [SP_PSI(His) 6 ] and three phytopathogenic fungi ( A. alternata , L. infectoria , and D. biseptata ). Untransformed K. lactis (strain GG799) and a K. lactis transformant expressing GFP were used as negative controls. The antifungal activity is given by the percentage of mycelial growth inhibition {MGI = [(free growth of the fungi − growth of the fungi in the presence of yeast)/(free growth of the fungi)] × 100}. All assays were performed in duplicate. (B) Representative plates showing antifungal activity toward A. alternata and L. infectoria . (C and D) Disk diffusion test. Impregnated disks with a purified sample of recombinant nonglycosylated cirsin PSI (0.47 mg/ml) were assayed against the fungus A. alternata by evaluating the appearance of a clear zone of inhibition. The assay was performed in triplicate at two different pH values (pH 5.0 and 6.8) using the antifungal amphotericin B (0.25 mg/ml) as a positive control. (C) Antifungal activity is represented by the percentage of mycelial growth inhibition {MGI = [(growth of the fungi in the presence of sample buffer − growth of the fungi in the presence of PSI)/(growth of fungi in the presence of sample buffer)] × 100}. (D) Representative plates showing antifungal activity of PSI toward A. alternata . The error bars indicate standard deviations.

    Techniques Used: Recombinant, Activity Assay, Expressing, Inhibition, Diffusion-based Assay, Purification, Positive Control

    4) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Rapid and parallel cloning using LIC-compatible expression vectors. Only two PCR fragments, one with and one without stop codon, are needed per target open reading frame for rapid and parallel insertion into ten LIC-compatible vectors. The vectors allow facile protein expression in four different hosts, i.e. E. coli , K. lactis , P. pastoris and L. tarentolae , and by in vitro transcription/translation. Vectors were constructed to support production of N- and C-terminal fusions to the IFP- and 6xHis-tags. The IFP moiety enables detection of IFP fusion proteins by easy-to-handle in-cell and in-gel infrared imaging, and the 6xHis-tag allows immunological detection of fusion proteins and affinity purification. A TEV protease cleavage site (not indicated) allows removal of the IFP- and 6xHis-tags. Photographs provided by: Wikipedia ( E. coli ); Linda Silveira, University of Redlands, California, USA ( K. lactis ); Dennis Kunkel, Dennis Kunkel Microscopy, Inc., Hawaii, USA ( P. pastoris ); Jena Bioscience, Jena, Germany ( L. tarentolae ).
    Figure Legend Snippet: Rapid and parallel cloning using LIC-compatible expression vectors. Only two PCR fragments, one with and one without stop codon, are needed per target open reading frame for rapid and parallel insertion into ten LIC-compatible vectors. The vectors allow facile protein expression in four different hosts, i.e. E. coli , K. lactis , P. pastoris and L. tarentolae , and by in vitro transcription/translation. Vectors were constructed to support production of N- and C-terminal fusions to the IFP- and 6xHis-tags. The IFP moiety enables detection of IFP fusion proteins by easy-to-handle in-cell and in-gel infrared imaging, and the 6xHis-tag allows immunological detection of fusion proteins and affinity purification. A TEV protease cleavage site (not indicated) allows removal of the IFP- and 6xHis-tags. Photographs provided by: Wikipedia ( E. coli ); Linda Silveira, University of Redlands, California, USA ( K. lactis ); Dennis Kunkel, Dennis Kunkel Microscopy, Inc., Hawaii, USA ( P. pastoris ); Jena Bioscience, Jena, Germany ( L. tarentolae ).

    Techniques Used: Clone Assay, Expressing, Polymerase Chain Reaction, In Vitro, Construct, Imaging, Affinity Purification, Microscopy

    LIC-compatible expression vectors. The pool of LIC-compatible vectors comprises vectors for expression of IFP fusion proteins in multiple expression systems, i.e. in vitro (LIC-pIVEX-LC1/-LC2) and in vivo in E. coli (LIC-pDEST-LC1/-LC2), K. lactis (LIC-pKLAC-LC1/-LC2), P. pastoris (LIC-pPICZ-LC1/-LC2) and L. tarentolae (LIC-pLEXSY-LC1/-LC2). The pool includes LC1 and LC2 vectors encoding 6xHis-IFP-TEV-ProteinX and ProteinX-TEV-IFP-6xHis fusions, respectively, after ligation-independent cloning of ProteinX-encoding open reading frames into the LCA and LCB sites. The maker proteins IFP and 6xHis can be cleaved off at the TEV protease cleavage site next to a PmeI site used for LIC. LCA and LCB sites are introduced into target open reading frames by PCR. The LIC fragment was designed on the basis of a 670-bp stuffer fragment, flanked by the LIC annealing sites LCA and LCB, respectively, both of which encompass a PmeI restriction site. The stop codon of LC2 vectors is provided by the vector whilst for LC1 vectors it has to be added by the reverse primer during PCR amplification of the target open reading frame.
    Figure Legend Snippet: LIC-compatible expression vectors. The pool of LIC-compatible vectors comprises vectors for expression of IFP fusion proteins in multiple expression systems, i.e. in vitro (LIC-pIVEX-LC1/-LC2) and in vivo in E. coli (LIC-pDEST-LC1/-LC2), K. lactis (LIC-pKLAC-LC1/-LC2), P. pastoris (LIC-pPICZ-LC1/-LC2) and L. tarentolae (LIC-pLEXSY-LC1/-LC2). The pool includes LC1 and LC2 vectors encoding 6xHis-IFP-TEV-ProteinX and ProteinX-TEV-IFP-6xHis fusions, respectively, after ligation-independent cloning of ProteinX-encoding open reading frames into the LCA and LCB sites. The maker proteins IFP and 6xHis can be cleaved off at the TEV protease cleavage site next to a PmeI site used for LIC. LCA and LCB sites are introduced into target open reading frames by PCR. The LIC fragment was designed on the basis of a 670-bp stuffer fragment, flanked by the LIC annealing sites LCA and LCB, respectively, both of which encompass a PmeI restriction site. The stop codon of LC2 vectors is provided by the vector whilst for LC1 vectors it has to be added by the reverse primer during PCR amplification of the target open reading frame.

    Techniques Used: Expressing, In Vitro, In Vivo, Ligation, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Amplification

    5) Product Images from "Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing"

    Article Title: Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2020.607507

    Chromatograms showing FOS catalytic products in 100 μL cell-free crude enzyme solution of K. lactis GG799 and GG799Δ Inv with 600 g L −1 sucrose solution for 2 h at 70°C. The peaks are annotated as follows: A = fructose, B = glucose, C = sucrose, D = neokestose, E = 1-kestose, F = 6-kestose, G = nystose, and H = 1 F -fructofranosylnystose.
    Figure Legend Snippet: Chromatograms showing FOS catalytic products in 100 μL cell-free crude enzyme solution of K. lactis GG799 and GG799Δ Inv with 600 g L −1 sucrose solution for 2 h at 70°C. The peaks are annotated as follows: A = fructose, B = glucose, C = sucrose, D = neokestose, E = 1-kestose, F = 6-kestose, G = nystose, and H = 1 F -fructofranosylnystose.

    Techniques Used:

    Time course of the online measured dissolved oxygen (DO) concentration and cell dry weight (CDW) in a fed-batch process using the Labfors3 system. (A) Production strain K. lactis GG799 and (B) mutant strain K. lactis GG799Δ Inv .
    Figure Legend Snippet: Time course of the online measured dissolved oxygen (DO) concentration and cell dry weight (CDW) in a fed-batch process using the Labfors3 system. (A) Production strain K. lactis GG799 and (B) mutant strain K. lactis GG799Δ Inv .

    Techniques Used: Concentration Assay, Mutagenesis

    Related Articles

    Positron Emission Tomography:

    Article Title: Directed evolution for improved total secretory protein production in Escherichia coli
    Article Snippet: .. For epPCR of high mutagenic rate, the 50-μL PCR mixture contained 1× standard Taq reaction buffer (Mg-free), 7 mM MgCl2 , 0.05 mM MnCl2 , 0.2 mM dATP, 0.2 mM dGTP, 1 mM dTTP, 1 mM dCTP, 20 pmol OsmY-DirEv-F primer, 20 pmol OsmY-DirEv-R primer , 50 ng pET-24a(+)-OsmY-Tfu0937 and 1.25 U Taq DNA polymerase (New England Biolabs). .. For epPCR of low error rate, the 50-μL PCR mixture contained 1×standard Taq reaction buffer (Mg-free), 1.5 mM MgCl2 , 0.01 mM MnCl2 , 0.3 mM of each dNTP, 4.5 pmol OsmY-DirEv-F primer, 4.5 pmol OsmY-DirEv-R , 7.77 ng pET-24a(+)-OsmY-Tfu0937 and 1.25 U Taq DNA polymerase (New England Biolabs).

    In Vitro:

    Article Title: Functional Dissection of Naturally Occurring Amino Acid Substitutions in eIF4E That Confers Recessive Potyvirus Resistance in Plants [W]Functional Dissection of Naturally Occurring Amino Acid Substitutions in eIF4E That Confers Recessive Potyvirus Resistance in Plants [W] [OA]
    Article Snippet: .. After eIF4E protein was eluted using the extraction buffer containing 100 uM m7 GTP, SDS-PAGE and immunoblot analysis were performed using a Capsicum -eIF4E antibody (New England Biolabs) for detection of eIF4E maintaining cap binding ability in vitro. .. The vector construction and tomato transformation followed previously described methods ( ).

    other:

    Article Title: N- and O-linked glycosylation of total plasma glycoproteins in galactosemia
    Article Snippet: Peptide N-glycosidase F (PNGase F), including denaturation buffer, digestion buffer, and NP-40 buffer were all purchased from New England Biolabs (Ipswich, MA).

    Article Title: Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2?-deoxyadenosine
    Article Snippet: Taq, Klenow fragment, Klenow fragment (exo– ), Bst, T7, GB-D (Deep Vent), GB-D (exo– ) [Deep Vent (exo– )], Tli (Vent) and Tli (exo– ) [Vent (exo– )] polymerases were purchased from New England Biolabs.

    Polymerase Chain Reaction:

    Article Title: Partitioning of Respiration in an Animal-Algal Symbiosis: Implications for Different Aerobic Capacity between Symbiodinium spp.
    Article Snippet: .. Primer validation was undertaken using end-point PCR, with each reaction containing 20 ng anemone DNA in a 20-μL mix of 1 × Standard Mg-free PCR buffer, 0.25 U Taq DNA polymerase, 0.25 μM (CO1 and ATP6 ) or 0.5 μM primers (EF-1- α), 2.5 mM MgCl2, and 0.25 mM dNTPs (reagents from New England Biolabs, Ipswich, MA, USA; primers from Integrated DNA Technologies, see above). .. Cycling conditions were 94°C for 2 min, followed by thirty cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 30 s, with a final elongation at 72°C for 10 min. Agarose gel electrophoresis confirmed a single PCR product for each primer set and, after product purification (ExoSAP-IT, Affymetrix, Santa Clara, CA, USA), amplicons were sequenced in both directions using the respective PCR primers (GeneWiz, South Plainfield, NJ, USA).

    Article Title: Directed evolution for improved total secretory protein production in Escherichia coli
    Article Snippet: .. For epPCR of high mutagenic rate, the 50-μL PCR mixture contained 1× standard Taq reaction buffer (Mg-free), 7 mM MgCl2 , 0.05 mM MnCl2 , 0.2 mM dATP, 0.2 mM dGTP, 1 mM dTTP, 1 mM dCTP, 20 pmol OsmY-DirEv-F primer, 20 pmol OsmY-DirEv-R primer , 50 ng pET-24a(+)-OsmY-Tfu0937 and 1.25 U Taq DNA polymerase (New England Biolabs). .. For epPCR of low error rate, the 50-μL PCR mixture contained 1×standard Taq reaction buffer (Mg-free), 1.5 mM MgCl2 , 0.01 mM MnCl2 , 0.3 mM of each dNTP, 4.5 pmol OsmY-DirEv-F primer, 4.5 pmol OsmY-DirEv-R , 7.77 ng pET-24a(+)-OsmY-Tfu0937 and 1.25 U Taq DNA polymerase (New England Biolabs).

    Article Title: A PCR-Based Method for Distinguishing between Two Common Beehive Bacteria, Paenibacillus larvae and Brevibacillus laterosporus
    Article Snippet: .. Template DNA for the PCR was extracted by adding part of a colony to 50 μl of distilled or deionized water in a PCR tube and boiling at 100°C for 10 min. TAQ (New England BioLabs) PCR was performed according to the manufacturer's instructions using the primers listed in . ..

    Binding Assay:

    Article Title: Functional Dissection of Naturally Occurring Amino Acid Substitutions in eIF4E That Confers Recessive Potyvirus Resistance in Plants [W]Functional Dissection of Naturally Occurring Amino Acid Substitutions in eIF4E That Confers Recessive Potyvirus Resistance in Plants [W] [OA]
    Article Snippet: .. After eIF4E protein was eluted using the extraction buffer containing 100 uM m7 GTP, SDS-PAGE and immunoblot analysis were performed using a Capsicum -eIF4E antibody (New England Biolabs) for detection of eIF4E maintaining cap binding ability in vitro. .. The vector construction and tomato transformation followed previously described methods ( ).

    SDS Page:

    Article Title: Functional Dissection of Naturally Occurring Amino Acid Substitutions in eIF4E That Confers Recessive Potyvirus Resistance in Plants [W]Functional Dissection of Naturally Occurring Amino Acid Substitutions in eIF4E That Confers Recessive Potyvirus Resistance in Plants [W] [OA]
    Article Snippet: .. After eIF4E protein was eluted using the extraction buffer containing 100 uM m7 GTP, SDS-PAGE and immunoblot analysis were performed using a Capsicum -eIF4E antibody (New England Biolabs) for detection of eIF4E maintaining cap binding ability in vitro. .. The vector construction and tomato transformation followed previously described methods ( ).

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    New England Biolabs k lactis protein expression kit
    Cirsin <t>PSI</t> fused to the α-MF leader sequence is efficiently integrated into the K. <t>lactis</t> genome and secreted into culture medium. (A) Schematic representation of the α-MF_PSI(His) 6 (top) and α-MF_PSI(N86S)(His) 6 (bottom) fusion proteins. Both sequences comprise the α-MF preprodomain sequence and a hexahistidine tag. The two putative N-glycosylation sites in the α-MF leader sequence (NGT and NTT) are indicated. A putative N-glycosylation site (NET) is present in the α-MF_PSI(His) 6 sequence, while in the construct α-MF_PSI(N86S)(His) 6 , this glycosylation site is mutated (N86S). (B) Confirmation by PCR of integration of the expression cassettes into the K. lactis genome. The numbers correspond to the selected clones and “c” to the amplification product using genomic DNA from untransformed K. lactis strain GG799 (negative control). (Top) α-MF_PSI(His) 6 . (Bottom) α-MF_PSI(N86S)(His) 6 . (C) Western blot analysis of recombinant cirsin PSI expression/secretion into culture media with an anti-His antibody. The numbers above the gels correspond to selected clones shown in panel B, and the lane marked with a c corresponds to the analysis of the culture medium of untransformed K. lactis GG799 cells (negative control). (Left) α-MF_PSI(His) 6 transformants. (Right) α-MF_PSI(N86S)(His) 6 transformants.
    K Lactis Protein Expression Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cirsin PSI fused to the α-MF leader sequence is efficiently integrated into the K. lactis genome and secreted into culture medium. (A) Schematic representation of the α-MF_PSI(His) 6 (top) and α-MF_PSI(N86S)(His) 6 (bottom) fusion proteins. Both sequences comprise the α-MF preprodomain sequence and a hexahistidine tag. The two putative N-glycosylation sites in the α-MF leader sequence (NGT and NTT) are indicated. A putative N-glycosylation site (NET) is present in the α-MF_PSI(His) 6 sequence, while in the construct α-MF_PSI(N86S)(His) 6 , this glycosylation site is mutated (N86S). (B) Confirmation by PCR of integration of the expression cassettes into the K. lactis genome. The numbers correspond to the selected clones and “c” to the amplification product using genomic DNA from untransformed K. lactis strain GG799 (negative control). (Top) α-MF_PSI(His) 6 . (Bottom) α-MF_PSI(N86S)(His) 6 . (C) Western blot analysis of recombinant cirsin PSI expression/secretion into culture media with an anti-His antibody. The numbers above the gels correspond to selected clones shown in panel B, and the lane marked with a c corresponds to the analysis of the culture medium of untransformed K. lactis GG799 cells (negative control). (Left) α-MF_PSI(His) 6 transformants. (Right) α-MF_PSI(N86S)(His) 6 transformants.

    Journal: Applied and Environmental Microbiology

    Article Title: Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

    doi: 10.1128/AEM.03151-13

    Figure Lengend Snippet: Cirsin PSI fused to the α-MF leader sequence is efficiently integrated into the K. lactis genome and secreted into culture medium. (A) Schematic representation of the α-MF_PSI(His) 6 (top) and α-MF_PSI(N86S)(His) 6 (bottom) fusion proteins. Both sequences comprise the α-MF preprodomain sequence and a hexahistidine tag. The two putative N-glycosylation sites in the α-MF leader sequence (NGT and NTT) are indicated. A putative N-glycosylation site (NET) is present in the α-MF_PSI(His) 6 sequence, while in the construct α-MF_PSI(N86S)(His) 6 , this glycosylation site is mutated (N86S). (B) Confirmation by PCR of integration of the expression cassettes into the K. lactis genome. The numbers correspond to the selected clones and “c” to the amplification product using genomic DNA from untransformed K. lactis strain GG799 (negative control). (Top) α-MF_PSI(His) 6 . (Bottom) α-MF_PSI(N86S)(His) 6 . (C) Western blot analysis of recombinant cirsin PSI expression/secretion into culture media with an anti-His antibody. The numbers above the gels correspond to selected clones shown in panel B, and the lane marked with a c corresponds to the analysis of the culture medium of untransformed K. lactis GG799 cells (negative control). (Left) α-MF_PSI(His) 6 transformants. (Right) α-MF_PSI(N86S)(His) 6 transformants.

    Article Snippet: Before yeast transformation, expression cassettes were obtained by linearizing all pKLAC1-PSI constructs with SacII, according to the manufacturers' instructions ( K. lactis Protein Expression kit; New England BioLabs).

    Techniques: Sequencing, Construct, Polymerase Chain Reaction, Expressing, Clone Assay, Amplification, Negative Control, Western Blot, Recombinant

    Recombinant cirsin PSI displays antifungal activity. (A) Confrontation assays between the selected K. lactis transformant secreting wt PSI [SP_PSI(His) 6 ] and three phytopathogenic fungi ( A. alternata , L. infectoria , and D. biseptata ). Untransformed K. lactis (strain GG799) and a K. lactis transformant expressing GFP were used as negative controls. The antifungal activity is given by the percentage of mycelial growth inhibition {MGI = [(free growth of the fungi − growth of the fungi in the presence of yeast)/(free growth of the fungi)] × 100}. All assays were performed in duplicate. (B) Representative plates showing antifungal activity toward A. alternata and L. infectoria . (C and D) Disk diffusion test. Impregnated disks with a purified sample of recombinant nonglycosylated cirsin PSI (0.47 mg/ml) were assayed against the fungus A. alternata by evaluating the appearance of a clear zone of inhibition. The assay was performed in triplicate at two different pH values (pH 5.0 and 6.8) using the antifungal amphotericin B (0.25 mg/ml) as a positive control. (C) Antifungal activity is represented by the percentage of mycelial growth inhibition {MGI = [(growth of the fungi in the presence of sample buffer − growth of the fungi in the presence of PSI)/(growth of fungi in the presence of sample buffer)] × 100}. (D) Representative plates showing antifungal activity of PSI toward A. alternata . The error bars indicate standard deviations.

    Journal: Applied and Environmental Microbiology

    Article Title: Establishing the Yeast Kluyveromyces lactis as an Expression Host for Production of the Saposin-Like Domain of the Aspartic Protease Cirsin

    doi: 10.1128/AEM.03151-13

    Figure Lengend Snippet: Recombinant cirsin PSI displays antifungal activity. (A) Confrontation assays between the selected K. lactis transformant secreting wt PSI [SP_PSI(His) 6 ] and three phytopathogenic fungi ( A. alternata , L. infectoria , and D. biseptata ). Untransformed K. lactis (strain GG799) and a K. lactis transformant expressing GFP were used as negative controls. The antifungal activity is given by the percentage of mycelial growth inhibition {MGI = [(free growth of the fungi − growth of the fungi in the presence of yeast)/(free growth of the fungi)] × 100}. All assays were performed in duplicate. (B) Representative plates showing antifungal activity toward A. alternata and L. infectoria . (C and D) Disk diffusion test. Impregnated disks with a purified sample of recombinant nonglycosylated cirsin PSI (0.47 mg/ml) were assayed against the fungus A. alternata by evaluating the appearance of a clear zone of inhibition. The assay was performed in triplicate at two different pH values (pH 5.0 and 6.8) using the antifungal amphotericin B (0.25 mg/ml) as a positive control. (C) Antifungal activity is represented by the percentage of mycelial growth inhibition {MGI = [(growth of the fungi in the presence of sample buffer − growth of the fungi in the presence of PSI)/(growth of fungi in the presence of sample buffer)] × 100}. (D) Representative plates showing antifungal activity of PSI toward A. alternata . The error bars indicate standard deviations.

    Article Snippet: Before yeast transformation, expression cassettes were obtained by linearizing all pKLAC1-PSI constructs with SacII, according to the manufacturers' instructions ( K. lactis Protein Expression kit; New England BioLabs).

    Techniques: Recombinant, Activity Assay, Expressing, Inhibition, Diffusion-based Assay, Purification, Positive Control

    Rapid and parallel cloning using LIC-compatible expression vectors. Only two PCR fragments, one with and one without stop codon, are needed per target open reading frame for rapid and parallel insertion into ten LIC-compatible vectors. The vectors allow facile protein expression in four different hosts, i.e. E. coli , K. lactis , P. pastoris and L. tarentolae , and by in vitro transcription/translation. Vectors were constructed to support production of N- and C-terminal fusions to the IFP- and 6xHis-tags. The IFP moiety enables detection of IFP fusion proteins by easy-to-handle in-cell and in-gel infrared imaging, and the 6xHis-tag allows immunological detection of fusion proteins and affinity purification. A TEV protease cleavage site (not indicated) allows removal of the IFP- and 6xHis-tags. Photographs provided by: Wikipedia ( E. coli ); Linda Silveira, University of Redlands, California, USA ( K. lactis ); Dennis Kunkel, Dennis Kunkel Microscopy, Inc., Hawaii, USA ( P. pastoris ); Jena Bioscience, Jena, Germany ( L. tarentolae ).

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Rapid and parallel cloning using LIC-compatible expression vectors. Only two PCR fragments, one with and one without stop codon, are needed per target open reading frame for rapid and parallel insertion into ten LIC-compatible vectors. The vectors allow facile protein expression in four different hosts, i.e. E. coli , K. lactis , P. pastoris and L. tarentolae , and by in vitro transcription/translation. Vectors were constructed to support production of N- and C-terminal fusions to the IFP- and 6xHis-tags. The IFP moiety enables detection of IFP fusion proteins by easy-to-handle in-cell and in-gel infrared imaging, and the 6xHis-tag allows immunological detection of fusion proteins and affinity purification. A TEV protease cleavage site (not indicated) allows removal of the IFP- and 6xHis-tags. Photographs provided by: Wikipedia ( E. coli ); Linda Silveira, University of Redlands, California, USA ( K. lactis ); Dennis Kunkel, Dennis Kunkel Microscopy, Inc., Hawaii, USA ( P. pastoris ); Jena Bioscience, Jena, Germany ( L. tarentolae ).

    Article Snippet: Protein expression in K. lactis LIC-pKLAC-LC1/LC2 plasmid templates were used for protein expression in K. lactis using the K. lactis Protein Expression Kit (NEB) as described in the manufacturer's instructions.

    Techniques: Clone Assay, Expressing, Polymerase Chain Reaction, In Vitro, Construct, Imaging, Affinity Purification, Microscopy

    LIC-compatible expression vectors. The pool of LIC-compatible vectors comprises vectors for expression of IFP fusion proteins in multiple expression systems, i.e. in vitro (LIC-pIVEX-LC1/-LC2) and in vivo in E. coli (LIC-pDEST-LC1/-LC2), K. lactis (LIC-pKLAC-LC1/-LC2), P. pastoris (LIC-pPICZ-LC1/-LC2) and L. tarentolae (LIC-pLEXSY-LC1/-LC2). The pool includes LC1 and LC2 vectors encoding 6xHis-IFP-TEV-ProteinX and ProteinX-TEV-IFP-6xHis fusions, respectively, after ligation-independent cloning of ProteinX-encoding open reading frames into the LCA and LCB sites. The maker proteins IFP and 6xHis can be cleaved off at the TEV protease cleavage site next to a PmeI site used for LIC. LCA and LCB sites are introduced into target open reading frames by PCR. The LIC fragment was designed on the basis of a 670-bp stuffer fragment, flanked by the LIC annealing sites LCA and LCB, respectively, both of which encompass a PmeI restriction site. The stop codon of LC2 vectors is provided by the vector whilst for LC1 vectors it has to be added by the reverse primer during PCR amplification of the target open reading frame.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: LIC-compatible expression vectors. The pool of LIC-compatible vectors comprises vectors for expression of IFP fusion proteins in multiple expression systems, i.e. in vitro (LIC-pIVEX-LC1/-LC2) and in vivo in E. coli (LIC-pDEST-LC1/-LC2), K. lactis (LIC-pKLAC-LC1/-LC2), P. pastoris (LIC-pPICZ-LC1/-LC2) and L. tarentolae (LIC-pLEXSY-LC1/-LC2). The pool includes LC1 and LC2 vectors encoding 6xHis-IFP-TEV-ProteinX and ProteinX-TEV-IFP-6xHis fusions, respectively, after ligation-independent cloning of ProteinX-encoding open reading frames into the LCA and LCB sites. The maker proteins IFP and 6xHis can be cleaved off at the TEV protease cleavage site next to a PmeI site used for LIC. LCA and LCB sites are introduced into target open reading frames by PCR. The LIC fragment was designed on the basis of a 670-bp stuffer fragment, flanked by the LIC annealing sites LCA and LCB, respectively, both of which encompass a PmeI restriction site. The stop codon of LC2 vectors is provided by the vector whilst for LC1 vectors it has to be added by the reverse primer during PCR amplification of the target open reading frame.

    Article Snippet: Protein expression in K. lactis LIC-pKLAC-LC1/LC2 plasmid templates were used for protein expression in K. lactis using the K. lactis Protein Expression Kit (NEB) as described in the manufacturer's instructions.

    Techniques: Expressing, In Vitro, In Vivo, Ligation, Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Amplification

    Chromatograms showing FOS catalytic products in 100 μL cell-free crude enzyme solution of K. lactis GG799 and GG799Δ Inv with 600 g L −1 sucrose solution for 2 h at 70°C. The peaks are annotated as follows: A = fructose, B = glucose, C = sucrose, D = neokestose, E = 1-kestose, F = 6-kestose, G = nystose, and H = 1 F -fructofranosylnystose.

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing

    doi: 10.3389/fbioe.2020.607507

    Figure Lengend Snippet: Chromatograms showing FOS catalytic products in 100 μL cell-free crude enzyme solution of K. lactis GG799 and GG799Δ Inv with 600 g L −1 sucrose solution for 2 h at 70°C. The peaks are annotated as follows: A = fructose, B = glucose, C = sucrose, D = neokestose, E = 1-kestose, F = 6-kestose, G = nystose, and H = 1 F -fructofranosylnystose.

    Article Snippet: K. lactis Production Mutant The K. lactis GG799 production host was generated as previously described (Spohner and Czermak, ) by introducing an FFT gene from A. terreus NIH2624 into the K. lactis GG799 wild-type strain from the K. lactis Protein Expression Kit (New England Biolabs, Frankfurt, Germany).

    Techniques:

    Time course of the online measured dissolved oxygen (DO) concentration and cell dry weight (CDW) in a fed-batch process using the Labfors3 system. (A) Production strain K. lactis GG799 and (B) mutant strain K. lactis GG799Δ Inv .

    Journal: Frontiers in Bioengineering and Biotechnology

    Article Title: Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing

    doi: 10.3389/fbioe.2020.607507

    Figure Lengend Snippet: Time course of the online measured dissolved oxygen (DO) concentration and cell dry weight (CDW) in a fed-batch process using the Labfors3 system. (A) Production strain K. lactis GG799 and (B) mutant strain K. lactis GG799Δ Inv .

    Article Snippet: K. lactis Production Mutant The K. lactis GG799 production host was generated as previously described (Spohner and Czermak, ) by introducing an FFT gene from A. terreus NIH2624 into the K. lactis GG799 wild-type strain from the K. lactis Protein Expression Kit (New England Biolabs, Frankfurt, Germany).

    Techniques: Concentration Assay, Mutagenesis