Structured Review

GE Healthcare pgex 4t3
Concept of the asymmetric directional T-vector. ( A ) Construction of <t>pGEX-4T3-PRESAT</t> and direct cloning of PCR product in pGEX-4T3-PRESAT. ( B ) The schematic representation of the ORF selection method using potential restriction enzyme site. The figure illustrates the case in which NcoI is chosen as the second restriction enzyme for selection. The rear PCR primer is designed with 5′-GG at the 5′ end, so that only the ligated plasmid with insert in the reverse orientation will have the NcoI site at the TA-cloning position. For NdeI selection, the rear primer with 5′-ATG is used instead of the 5′-GG primer.
Pgex 4t3, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pgex 4t3/product/GE Healthcare
Average 99 stars, based on 27 article reviews
Price from $9.99 to $1999.99
pgex 4t3 - by Bioz Stars, 2020-02
99/100 stars

Images

1) Product Images from "The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics"

Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics

Journal: Protein Science : A Publication of the Protein Society

doi: 10.1110/ps.03439004

Concept of the asymmetric directional T-vector. ( A ) Construction of pGEX-4T3-PRESAT and direct cloning of PCR product in pGEX-4T3-PRESAT. ( B ) The schematic representation of the ORF selection method using potential restriction enzyme site. The figure illustrates the case in which NcoI is chosen as the second restriction enzyme for selection. The rear PCR primer is designed with 5′-GG at the 5′ end, so that only the ligated plasmid with insert in the reverse orientation will have the NcoI site at the TA-cloning position. For NdeI selection, the rear primer with 5′-ATG is used instead of the 5′-GG primer.
Figure Legend Snippet: Concept of the asymmetric directional T-vector. ( A ) Construction of pGEX-4T3-PRESAT and direct cloning of PCR product in pGEX-4T3-PRESAT. ( B ) The schematic representation of the ORF selection method using potential restriction enzyme site. The figure illustrates the case in which NcoI is chosen as the second restriction enzyme for selection. The rear PCR primer is designed with 5′-GG at the 5′ end, so that only the ligated plasmid with insert in the reverse orientation will have the NcoI site at the TA-cloning position. For NdeI selection, the rear primer with 5′-ATG is used instead of the 5′-GG primer.

Techniques Used: Plasmid Preparation, Clone Assay, Polymerase Chain Reaction, Selection, TA Cloning

Concept of HTS for the soluble GST-fusion proteins from protein expression libraries. (i) Various PCR primers were designed and used to amplify a series of partial cDNA fragments of the protein of interest, which may contain a putative domain. (ii) The fragments are inserted into pGEX-4T3-PRESAT vector, selected for ORF orientation by restriction cleavage, and transformed into E. coli for colony isolation. (iii) Deep 96-well format plates are used to culture individual clones to allow expression of the GST-fusion proteins. (iv) The clone displaying the highest CDNB assay signal will be selected for further analysis.
Figure Legend Snippet: Concept of HTS for the soluble GST-fusion proteins from protein expression libraries. (i) Various PCR primers were designed and used to amplify a series of partial cDNA fragments of the protein of interest, which may contain a putative domain. (ii) The fragments are inserted into pGEX-4T3-PRESAT vector, selected for ORF orientation by restriction cleavage, and transformed into E. coli for colony isolation. (iii) Deep 96-well format plates are used to culture individual clones to allow expression of the GST-fusion proteins. (iv) The clone displaying the highest CDNB assay signal will be selected for further analysis.

Techniques Used: Expressing, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay, Isolation, Clone Assay

2) Product Images from "Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿"

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿

Journal: Clinical and Vaccine Immunology : CVI

doi: 10.1128/CVI.00319-08

Production and confirmation of antigenicity of TCoV N recombinant fusion protein produced by E. coli BL-21 transfected with pGEX-4T3-TCoV-N using 12% discontinuous SDS-PAGE (lanes 1 to 3) and subsequent Western blotting with anti-GST and anti-TCoV N-protein MAbs 1.01 and 4.23 (lanes 4 to 7). Lane M, molecular mass marker in kilodaltons; lane 1, nonpurified bacterial lysate after induction with IPTG; lane 2, TCoV N fusion protein purified with GST beads from bacterial lysate after induction with IPTG; lane 3, the same as lane 2 but from noninduced bacterial culture; lanes 4 and 5, Western blot using anti-GST antibodies against the TCoV N fusion protein expressed and purified as in lanes 2 and 3, respectively; lanes 6 and 7, Western blot using MAbs 1.01 and 4.23, respectively, against the TCoV N fusion protein expressed and purified as described for lane 2.
Figure Legend Snippet: Production and confirmation of antigenicity of TCoV N recombinant fusion protein produced by E. coli BL-21 transfected with pGEX-4T3-TCoV-N using 12% discontinuous SDS-PAGE (lanes 1 to 3) and subsequent Western blotting with anti-GST and anti-TCoV N-protein MAbs 1.01 and 4.23 (lanes 4 to 7). Lane M, molecular mass marker in kilodaltons; lane 1, nonpurified bacterial lysate after induction with IPTG; lane 2, TCoV N fusion protein purified with GST beads from bacterial lysate after induction with IPTG; lane 3, the same as lane 2 but from noninduced bacterial culture; lanes 4 and 5, Western blot using anti-GST antibodies against the TCoV N fusion protein expressed and purified as in lanes 2 and 3, respectively; lanes 6 and 7, Western blot using MAbs 1.01 and 4.23, respectively, against the TCoV N fusion protein expressed and purified as described for lane 2.

Techniques Used: Recombinant, Produced, Transfection, SDS Page, Western Blot, Marker, Purification

Related Articles

Clone Assay:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: Paragraph title: Cloning of the TCoV N-protein genes. ... PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit.

Article Title:
Article Snippet: To create the plasmid expressing the 763-amino acid HA-tagged DYRK1A isoform (from hereon identified as +9), we cloned a BglII–SacI fragment (amino acids 1–208) from the plasmid pEGFP-DYRK1A (+9) and a SacI–XbaI fragment (amino acids 209–763) from the plasmid pHA-DYRK1A into the BamHI and XbaI sites of pCDNA-HA. .. Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: .. FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ). .. Alternatively, FLAG-Sprouty2 was subcloned into pGEX-6P3 (GE Healthcare), expressed and purified as described above.

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: FLAG-nsp1 of EAV and FLAG-nsp1γ of SHFV were also cloned into pXJ41. .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences.

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST. .. Plasmid pGEX-NMHC-IIA, encoding a fusion protein of GST and the rod domain of human NMHC-IIA, was described previously ( ) and used to generate GST-NMHC-IIA.

Article Title: MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression
Article Snippet: .. Molecular cloning MISP (DKFZp686H18209) was received from S. Wiemann (DKFZ, Heidelberg, Germany), PCR amplified and cloned into the XhoI–BamHI sites of the pEGFP-C1 (Takara Bio Inc.), the BamHI–XhoI sites of pGEX-4T3 (GE Healthcare), and of pCMV-3Tag-1 (Agilent Technologies). .. All MISP fragments were PCR amplified and cloned into the BamHI–XhoI sites of pCMV-3Tag-1. pRcCMV-Plk1 was received from S. Lev (Weizmann Institute, Rehovot, Israel), PCR amplified and cloned into the EcoRI–XhoI sites of pGEX-4T1 and the EcoRI–XhoI sites of pCMV-3Tag-1.

Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
Article Snippet: Oligonucleotide primers were obtained from Hokkaido System Science. pGEM-T (Promega), pGEX-4T3 (Amersham Bioscience), and pET32a (Novagen), respectively, were purchased. .. For PCR cloning, QUICK-Clone cDNA of mouse spleen and human HeLa cell (Clontech) were used as a template.

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden). .. Human SNX27 was also cloned by PCR using the same cDNAs to amplify PXK and subcloned into pEGFP-N1 vector.

Article Title: Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Article Snippet: Paragraph title: Plasmids and HIV-1 molecular clones. ... NL4-3GFPΔ Vpr was constructed by replacing the Nef coding sequence of the NL4-3GFPΔ Vpr obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, Md.) with the Nef-GFP fusion sequence of NL4-3GFP. pCDNA3-14-3-3η and pGEX-14-3-3η were constructed by introducing 14-3-3η cDNA into pCDNA3 (Invitrogen, Carlsbad, Calif.) and pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.), respectively. pB42AD-14-3-3η (1-244), (110-244), (141-244), (168-244), (190-210) and (210-244) were constructed by inserting 14-3-3η cDNA fragments of the indicated portions of 14-3-3η into pB42AD (Clontech). pGAD424-14-3-3σ (1-270) and (106-270) were constructed by subcloning 14-3-3σ cDNA fragments of 1-270 or 106-270 of 14-3-3σ into pGAD424 (Clontech), respectively.

Article Title: Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation
Article Snippet: .. GST pull down GST fusions were expressed from pGex-4T3 (Amersham) and untagged proteins were expressed from pRSF1b (Merck); in all cases full-length T. brucei RAD51 or RAD51 paralogue ORFs were cloned by PCR (primers available on request). .. Normally, Rosetta E. coli cells were transformed the GST fusion and untagged protein expression vectors to allow coexpression and GST pull down.

Amplification:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: The N gene of TCoV-MG10 was amplified using NBEF (5′ - AAA GGATCC ATGGCAAGCGGTAAGGCAAC-3′) and NBER (5′-AAA GAATTC CTCTACAACTCATTCTCTC-3′) primers containing restriction sites (underlined) for BamHI and EcoRI, respectively. .. PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit.

Article Title: MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression
Article Snippet: .. Molecular cloning MISP (DKFZp686H18209) was received from S. Wiemann (DKFZ, Heidelberg, Germany), PCR amplified and cloned into the XhoI–BamHI sites of the pEGFP-C1 (Takara Bio Inc.), the BamHI–XhoI sites of pGEX-4T3 (GE Healthcare), and of pCMV-3Tag-1 (Agilent Technologies). .. All MISP fragments were PCR amplified and cloned into the BamHI–XhoI sites of pCMV-3Tag-1. pRcCMV-Plk1 was received from S. Lev (Weizmann Institute, Rehovot, Israel), PCR amplified and cloned into the EcoRI–XhoI sites of pGEX-4T1 and the EcoRI–XhoI sites of pCMV-3Tag-1.

Article Title: GxcC connects Rap and Rac signaling during Dictyostelium development
Article Snippet: An N-terminal construct of GxcC (base pairs 1–1911) was amplified using the forward primer 5′ -GGATCCATGCCAATTCAATTTGATGC-3′ and reverse primer 5′ -GCTAGCTTCAGCTTCATCTTCATCATCTTC-3′ ). .. For expression in E . coli the digested N-terminal GxcC fragment was ligated into the BamHI site of pGEX 4T3 (GE Healthcare, N-terminal GST).

Construct:

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: pEF Myc-B-Raf constructs and pcDNA3 FLAG-Sprouty2 were described previously ( ). pcDNA3 HA B-Raf was from W. Kolch, Beatson Institute, UK. pEXV MEK:EE was from C. J. Marshall, Institute for Cancer Research, UK. .. FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ).

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST. .. Plasmid pGEX-NMHC-IIA, encoding a fusion protein of GST and the rod domain of human NMHC-IIA, was described previously ( ) and used to generate GST-NMHC-IIA.

Article Title: GxcC connects Rap and Rac signaling during Dictyostelium development
Article Snippet: A C-terminal GxcC (basepairs 2578–3648) construct was obtained with the primers: 5′ -GGATCCGATCAACAAGATAAAGCTTCAC3′ and 5′ GCTAGCTTATTTTTTTGAAGTTAAAACTTTTG-3′ ). .. For expression in E . coli the digested N-terminal GxcC fragment was ligated into the BamHI site of pGEX 4T3 (GE Healthcare, N-terminal GST).

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: Paragraph title: Molecular cloning of human PXK , preparation of expression constructs, and site-directed mutagenesis. ... The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden).

Article Title: Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Article Snippet: .. NL4-3GFPΔ Vpr was constructed by replacing the Nef coding sequence of the NL4-3GFPΔ Vpr obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, Md.) with the Nef-GFP fusion sequence of NL4-3GFP. pCDNA3-14-3-3η and pGEX-14-3-3η were constructed by introducing 14-3-3η cDNA into pCDNA3 (Invitrogen, Carlsbad, Calif.) and pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.), respectively. pB42AD-14-3-3η (1-244), (110-244), (141-244), (168-244), (190-210) and (210-244) were constructed by inserting 14-3-3η cDNA fragments of the indicated portions of 14-3-3η into pB42AD (Clontech). pGAD424-14-3-3σ (1-270) and (106-270) were constructed by subcloning 14-3-3σ cDNA fragments of 1-270 or 106-270 of 14-3-3σ into pGAD424 (Clontech), respectively. .. Myc epitope-tagged Cdc25C-expressing plasmids, Myc-Cdc25C WT and S216A mutant, were kind gifts from J.

Article Title: Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains *
Article Snippet: To express and purify recombinant Nox4 and Nox2 dehydrogenase domain proteins, constructs were created starting from the end of the last predicted transmembrane helix to the C terminus consisting of residues Nox4-(304–578) (GenBankTM accession number ) and Nox2-(290–570) (GenBankTM accession number ) for full-length dehydrogenase domains. .. All PCR products were digested with BamHI and SalI and ligated into pGEX 4T3 (GE Healthcare) to generate glutathione S -transferase (GST) fusion proteins.

Incubation:

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ). .. Flag Sprouty2 was cleaved from GST by incubation with 80 U Prescission Protease (GE Healthcare) in cleavage buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) for 4 h at 4°C.

Luciferase:

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: Plasmids pCAGT7, encoding T7 RNA polymerase, and pT7EMCLuc, carrying the firefly luciferase gene under the control of the T7 promoter, were used to determine fusion efficiency as described previously ( ). .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST.

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Activity Assay:

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cell Culture:

Article Title: Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains *
Article Snippet: All PCR products were digested with BamHI and SalI and ligated into pGEX 4T3 (GE Healthcare) to generate glutathione S -transferase (GST) fusion proteins. .. Proteins were expressed in Escherichia coli BL21 DE3 pLys and cultured at 37 °C until reaching an A 600 ∼0.6–0.8.

Expressing:

Article Title:
Article Snippet: .. Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). .. To generate the GST-DYRK1A/S520A–expressing plasmid, a SacI–NotI fragment (amino acids 200–754) in pGST-DYRK1A was replaced by the equivalent fragment from pHA-DYRK1A/S520A, which contains the point mutation.

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus
Article Snippet: .. For expression of GFP in E. coli , the EGFP gene was subcloned from pEGFP-N1 (Clontech) at the Sma I-Nco I sites in pGEX-4T3 (Amersham Biosciences). .. A pET-N-Sac was derived from pET-N by introducing a Sac I restriction site at the end of the N-coding sequence by using the Pfu DNA polymerase with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions (sequence of primers available on demand).

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: Plasmids pPEP98-gB, pPEP99-gD, pPEP101-gL, and pPEP100-gH were used for expression of HSV-1 gB, gD, gL, and gH, respectively, as described previously ( ). .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST.

Article Title: GxcC connects Rap and Rac signaling during Dictyostelium development
Article Snippet: .. For expression in E . coli the digested N-terminal GxcC fragment was ligated into the BamHI site of pGEX 4T3 (GE Healthcare, N-terminal GST). .. The gxcC gene disruption construct was created by amplifying the GxcC fragment from position 1660 to 3648 (5′ -GGATCCAGAAATCGTAAGGTTATGAATG-3′ , 5′ -GCTAGCTTATTTTTTTGAAGTTAAAACTTTTG-3′ ).

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: .. The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden). .. Human SNX27 was also cloned by PCR using the same cDNAs to amplify PXK and subcloned into pEGFP-N1 vector.

Article Title: Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation
Article Snippet: GST pull down GST fusions were expressed from pGex-4T3 (Amersham) and untagged proteins were expressed from pRSF1b (Merck); in all cases full-length T. brucei RAD51 or RAD51 paralogue ORFs were cloned by PCR (primers available on request). .. Normally, Rosetta E. coli cells were transformed the GST fusion and untagged protein expression vectors to allow coexpression and GST pull down.

Transformation Assay:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit. .. Escherichia coli DH5α competent cells were transformed with the ligation mix and plated onto LB agar plates containing ampicillin.

Article Title: Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation
Article Snippet: GST pull down GST fusions were expressed from pGex-4T3 (Amersham) and untagged proteins were expressed from pRSF1b (Merck); in all cases full-length T. brucei RAD51 or RAD51 paralogue ORFs were cloned by PCR (primers available on request). .. Normally, Rosetta E. coli cells were transformed the GST fusion and untagged protein expression vectors to allow coexpression and GST pull down.

Over Expression:

Article Title: Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation
Article Snippet: GST pull down GST fusions were expressed from pGex-4T3 (Amersham) and untagged proteins were expressed from pRSF1b (Merck); in all cases full-length T. brucei RAD51 or RAD51 paralogue ORFs were cloned by PCR (primers available on request). .. In either case, a 50 ml culture of E. coli cells carrying the appropriate overexpression vectors, maintained by selection with antibiotics, were grown at 20°C until an OD of ∼ 0.5, at which time protein expression was induced with 1 mM IPTG overnight.

Derivative Assay:

Article Title: Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus
Article Snippet: For expression of GFP in E. coli , the EGFP gene was subcloned from pEGFP-N1 (Clontech) at the Sma I-Nco I sites in pGEX-4T3 (Amersham Biosciences). .. A pET-N-Sac was derived from pET-N by introducing a Sac I restriction site at the end of the N-coding sequence by using the Pfu DNA polymerase with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions (sequence of primers available on demand).

Gel Purification:

Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
Article Snippet: T4 DNA ligase, Wizard plasmid miniprep kit, and Wizard SV DNA gel purification kit (Promega) were used for ligation and plasmid purification. .. Oligonucleotide primers were obtained from Hokkaido System Science. pGEM-T (Promega), pGEX-4T3 (Amersham Bioscience), and pET32a (Novagen), respectively, were purchased.

Transfection:

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. DNA transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Ligation:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit. .. The ligation of the digested product with linearized pGEX-4T3 was performed using T4 DNA ligase (Invitrogen).

Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
Article Snippet: T4 DNA ligase, Wizard plasmid miniprep kit, and Wizard SV DNA gel purification kit (Promega) were used for ligation and plasmid purification. .. Oligonucleotide primers were obtained from Hokkaido System Science. pGEM-T (Promega), pGEX-4T3 (Amersham Bioscience), and pET32a (Novagen), respectively, were purchased.

Protease Inhibitor:

Article Title: Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains *
Article Snippet: All PCR products were digested with BamHI and SalI and ligated into pGEX 4T3 (GE Healthcare) to generate glutathione S -transferase (GST) fusion proteins. .. Cells were lysed in 50 m m Tris-HCl, pH 7.5, 150 m m NaCl, 10 μ m FAD, 1 m m phenylmethylsulfonyl fluoride, 100 μ m diisopropyl fluorophosphate, 0.05% (v/v) Tween 20, 1 m m 1,4-dithiothreitol, with Complete protease inhibitor mixture (Roche Applied Science).

Infection:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: The total RNA was extracted from the intestinal tissue homogenates of infected turkey poults using the Qiagen RNeasy kit (Qiagen, Valencia, CA). .. PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit.

Generated:

Article Title:
Article Snippet: .. Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). .. To generate the GST-DYRK1A/S520A–expressing plasmid, a SacI–NotI fragment (amino acids 200–754) in pGST-DYRK1A was replaced by the equivalent fragment from pHA-DYRK1A/S520A, which contains the point mutation.

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ). .. In vitro transcribed and translated (IVTT) [35 S]-Methionine-labeled HA B-Raf was generated from pcDNA3 HA B-Raf using the TNT T7 Quick Coupled Transcription Translation kit (Promega) according to the manufacturer's protocol.

Article Title: Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation
Article Snippet: .. GST-NIC (NIC1747-2444), GST-NICΔRAM (NIC1810-2444), GST-NICΔANK (NIC1747-1863 and 2077-2444), and GST-NICΔTAD (NIC1747-2193) were generated by subcloning NIC and its mutants into pGEX-4T3 (Amersham Pharmacia). .. Recombinant GST fusion proteins were then purified on GSH agarose.

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: .. The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden). .. Human SNX27 was also cloned by PCR using the same cDNAs to amplify PXK and subcloned into pEGFP-N1 vector.

DNA Sequencing:

Article Title:
Article Snippet: The plasmid pHA-DYRK1A/S520A and the kinase-inactive mutants pHA-DYRK1A/K179R and pHA-DYRK1A/Y310,312F were generated by site-directed mutagenesis and verified by DNA sequencing. .. Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

Sequencing:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit. .. Transformants were screened by digestion with BamHI and EcoRI, and clones containing inserts were checked for orientation by sequencing.

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus
Article Snippet: For expression of GFP in E. coli , the EGFP gene was subcloned from pEGFP-N1 (Clontech) at the Sma I-Nco I sites in pGEX-4T3 (Amersham Biosciences). .. A pET-N-Sac was derived from pET-N by introducing a Sac I restriction site at the end of the N-coding sequence by using the Pfu DNA polymerase with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions (sequence of primers available on demand).

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST. .. Plasmid pGEX-NMHC-IIA, encoding a fusion protein of GST and the rod domain of human NMHC-IIA, was described previously ( ) and used to generate GST-NMHC-IIA.

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: FLAG-mCLOCK, Myc-BMAL1, CMX-CLOCK, and CMX-MOP4 were kindly donated by Dr. P. Sassone-Corsi (University of California, Irvine, CA, USA) and Dr. Garret A. FitzGerald (University of Pennsylvania, Philadelphia, PA, USA; refs. , ). pVP16-CLOCK, which expresses CLOCK fused with the VP16 activation domain (AD), was constructed by subcloning the coding sequence of the mouse CLOCK into pVP16 (Clontech, Palo Alto, CA, USA) in an inframe fashion. .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega.

Article Title: Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Article Snippet: .. NL4-3GFPΔ Vpr was constructed by replacing the Nef coding sequence of the NL4-3GFPΔ Vpr obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, Md.) with the Nef-GFP fusion sequence of NL4-3GFP. pCDNA3-14-3-3η and pGEX-14-3-3η were constructed by introducing 14-3-3η cDNA into pCDNA3 (Invitrogen, Carlsbad, Calif.) and pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.), respectively. pB42AD-14-3-3η (1-244), (110-244), (141-244), (168-244), (190-210) and (210-244) were constructed by inserting 14-3-3η cDNA fragments of the indicated portions of 14-3-3η into pB42AD (Clontech). pGAD424-14-3-3σ (1-270) and (106-270) were constructed by subcloning 14-3-3σ cDNA fragments of 1-270 or 106-270 of 14-3-3σ into pGAD424 (Clontech), respectively. .. Myc epitope-tagged Cdc25C-expressing plasmids, Myc-Cdc25C WT and S216A mutant, were kind gifts from J.

Binding Assay:

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST. .. Plasmid pME-Fc-gB, used to generate an extracellular domain of gB (codons 31 to 730) fused to a mutant Fc fragment of human IgG1 with low binding affinity for cellular Fc receptors (gB-Fc), and plasmid pME-Fc-gD, used to generate an extracellular domain of gD (codons 31 to 336) fused to the mutant Fc fragment of human IgG1 (gD-Fc), were constructed as described earlier ( ). pME-Fc-CD200 ( ) was used to generate control Fc fusion protein.

Molecular Cloning:

Article Title: MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression
Article Snippet: .. Molecular cloning MISP (DKFZp686H18209) was received from S. Wiemann (DKFZ, Heidelberg, Germany), PCR amplified and cloned into the XhoI–BamHI sites of the pEGFP-C1 (Takara Bio Inc.), the BamHI–XhoI sites of pGEX-4T3 (GE Healthcare), and of pCMV-3Tag-1 (Agilent Technologies). .. All MISP fragments were PCR amplified and cloned into the BamHI–XhoI sites of pCMV-3Tag-1. pRcCMV-Plk1 was received from S. Lev (Weizmann Institute, Rehovot, Israel), PCR amplified and cloned into the EcoRI–XhoI sites of pGEX-4T1 and the EcoRI–XhoI sites of pCMV-3Tag-1.

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: Paragraph title: Molecular cloning of human PXK , preparation of expression constructs, and site-directed mutagenesis. ... The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden).

Pull Down Assay:

Article Title: Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation
Article Snippet: Paragraph title: GST pull-down assay ... GST-NIC (NIC1747-2444), GST-NICΔRAM (NIC1810-2444), GST-NICΔANK (NIC1747-1863 and 2077-2444), and GST-NICΔTAD (NIC1747-2193) were generated by subcloning NIC and its mutants into pGEX-4T3 (Amersham Pharmacia).

Mutagenesis:

Article Title:
Article Snippet: The plasmid pHA-DYRK1A/S520A and the kinase-inactive mutants pHA-DYRK1A/K179R and pHA-DYRK1A/Y310,312F were generated by site-directed mutagenesis and verified by DNA sequencing. .. Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: For site directed mutagenesis, FLAG-Sprouty2 was subcloned into the Gateway vector system (Invitrogen) and point mutations were introduced using the QuickChange kit (Stratagene) according to manufacturer's protocol. .. FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ).

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus
Article Snippet: For expression of GFP in E. coli , the EGFP gene was subcloned from pEGFP-N1 (Clontech) at the Sma I-Nco I sites in pGEX-4T3 (Amersham Biosciences). .. A pET-N-Sac was derived from pET-N by introducing a Sac I restriction site at the end of the N-coding sequence by using the Pfu DNA polymerase with the QuickChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions (sequence of primers available on demand).

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST. .. Plasmid pME-Fc-gB, used to generate an extracellular domain of gB (codons 31 to 730) fused to a mutant Fc fragment of human IgG1 with low binding affinity for cellular Fc receptors (gB-Fc), and plasmid pME-Fc-gD, used to generate an extracellular domain of gD (codons 31 to 336) fused to the mutant Fc fragment of human IgG1 (gD-Fc), were constructed as described earlier ( ). pME-Fc-CD200 ( ) was used to generate control Fc fusion protein.

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: Paragraph title: Molecular cloning of human PXK , preparation of expression constructs, and site-directed mutagenesis. ... The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden).

Article Title: Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Article Snippet: Vpr-related plasmids pLexA-VPR, pCMV-FLAG-VPR, pCDNA3-VPR, and pLexA-Tat were described previously ( ). pLexA-VPRL64A, VprL23A, VprL23,64,67A, and VprR80A were constructed by substituting the indicated amino acids of Vpr into the pLexA-VPR using a PCR-assisted in vitro mutagenesis reaction. pLexA-Vpr (64-96) was constructed by inserting the corresponding Vpr cDNA fragment into pLexA (Clontech, Palo Alto, Calif.). .. NL4-3GFPΔ Vpr was constructed by replacing the Nef coding sequence of the NL4-3GFPΔ Vpr obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, Md.) with the Nef-GFP fusion sequence of NL4-3GFP. pCDNA3-14-3-3η and pGEX-14-3-3η were constructed by introducing 14-3-3η cDNA into pCDNA3 (Invitrogen, Carlsbad, Calif.) and pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.), respectively. pB42AD-14-3-3η (1-244), (110-244), (141-244), (168-244), (190-210) and (210-244) were constructed by inserting 14-3-3η cDNA fragments of the indicated portions of 14-3-3η into pB42AD (Clontech). pGAD424-14-3-3σ (1-270) and (106-270) were constructed by subcloning 14-3-3σ cDNA fragments of 1-270 or 106-270 of 14-3-3σ into pGAD424 (Clontech), respectively.

Subcloning:

Article Title:
Article Snippet: .. Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). .. To generate the GST-DYRK1A/S520A–expressing plasmid, a SacI–NotI fragment (amino acids 200–754) in pGST-DYRK1A was replaced by the equivalent fragment from pHA-DYRK1A/S520A, which contains the point mutation.

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation
Article Snippet: .. GST-NIC (NIC1747-2444), GST-NICΔRAM (NIC1810-2444), GST-NICΔANK (NIC1747-1863 and 2077-2444), and GST-NICΔTAD (NIC1747-2193) were generated by subcloning NIC and its mutants into pGEX-4T3 (Amersham Pharmacia). .. Recombinant GST fusion proteins were then purified on GSH agarose.

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: .. The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden). .. Human SNX27 was also cloned by PCR using the same cDNAs to amplify PXK and subcloned into pEGFP-N1 vector.

Article Title: Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Article Snippet: .. NL4-3GFPΔ Vpr was constructed by replacing the Nef coding sequence of the NL4-3GFPΔ Vpr obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, Md.) with the Nef-GFP fusion sequence of NL4-3GFP. pCDNA3-14-3-3η and pGEX-14-3-3η were constructed by introducing 14-3-3η cDNA into pCDNA3 (Invitrogen, Carlsbad, Calif.) and pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.), respectively. pB42AD-14-3-3η (1-244), (110-244), (141-244), (168-244), (190-210) and (210-244) were constructed by inserting 14-3-3η cDNA fragments of the indicated portions of 14-3-3η into pB42AD (Clontech). pGAD424-14-3-3σ (1-270) and (106-270) were constructed by subcloning 14-3-3σ cDNA fragments of 1-270 or 106-270 of 14-3-3σ into pGAD424 (Clontech), respectively. .. Myc epitope-tagged Cdc25C-expressing plasmids, Myc-Cdc25C WT and S216A mutant, were kind gifts from J.

Purification:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: .. PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit. .. The ligation of the digested product with linearized pGEX-4T3 was performed using T4 DNA ligase (Invitrogen).

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: .. FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ). .. Alternatively, FLAG-Sprouty2 was subcloned into pGEX-6P3 (GE Healthcare), expressed and purified as described above.

Article Title: Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation
Article Snippet: GST-NIC (NIC1747-2444), GST-NICΔRAM (NIC1810-2444), GST-NICΔANK (NIC1747-1863 and 2077-2444), and GST-NICΔTAD (NIC1747-2193) were generated by subcloning NIC and its mutants into pGEX-4T3 (Amersham Pharmacia). .. Recombinant GST fusion proteins were then purified on GSH agarose.

Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
Article Snippet: T4 DNA ligase, Wizard plasmid miniprep kit, and Wizard SV DNA gel purification kit (Promega) were used for ligation and plasmid purification. .. Oligonucleotide primers were obtained from Hokkaido System Science. pGEM-T (Promega), pGEX-4T3 (Amersham Bioscience), and pET32a (Novagen), respectively, were purchased.

Protein Purification:

Article Title: Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains *
Article Snippet: Paragraph title: Recombinant Protein Purification ... All PCR products were digested with BamHI and SalI and ligated into pGEX 4T3 (GE Healthcare) to generate glutathione S -transferase (GST) fusion proteins.

Polymerase Chain Reaction:

Article Title: Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen ▿
Article Snippet: The PCR product was purified using the Mini Elute purification kit (Qiagen, Valencia, CA) and double-digested with BamHI and EcoRI (New England BioLabs, Ipswich, MA). .. PGEX-4T3 (Amersham Pharmacia Biotech, Sweden) was digested by both BamHI and EcoRI and dephosphorylated using shrimp alkaline phosphatase (AP; USB Corporation, Cleveland, OH) prior to purification with the Mini Elute purification kit.

Article Title:
Article Snippet: Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). .. Plasmid pEGFP-DYRK1A/1–167 was made by subcloning a BamHI–SalI PCR fragment (amino acids 1–167) into pEGFP BglII-XhoI.

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST. .. Plasmid pGEX-NMHC-IIA, encoding a fusion protein of GST and the rod domain of human NMHC-IIA, was described previously ( ) and used to generate GST-NMHC-IIA.

Article Title: MISP is a novel Plk1 substrate required for proper spindle orientation and mitotic progression
Article Snippet: .. Molecular cloning MISP (DKFZp686H18209) was received from S. Wiemann (DKFZ, Heidelberg, Germany), PCR amplified and cloned into the XhoI–BamHI sites of the pEGFP-C1 (Takara Bio Inc.), the BamHI–XhoI sites of pGEX-4T3 (GE Healthcare), and of pCMV-3Tag-1 (Agilent Technologies). .. All MISP fragments were PCR amplified and cloned into the BamHI–XhoI sites of pCMV-3Tag-1. pRcCMV-Plk1 was received from S. Lev (Weizmann Institute, Rehovot, Israel), PCR amplified and cloned into the EcoRI–XhoI sites of pGEX-4T1 and the EcoRI–XhoI sites of pCMV-3Tag-1.

Article Title: GxcC connects Rap and Rac signaling during Dictyostelium development
Article Snippet: The obtained PCR fragments were digested with BamHI and ligated into the BglII site of the previously characterized Dictyostelium extrachromosomal plasmids pDM314 (N-terminal GST) and pDM317 (N-terminal GFP) [ ]. .. For expression in E . coli the digested N-terminal GxcC fragment was ligated into the BamHI site of pGEX 4T3 (GE Healthcare, N-terminal GST).

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
Article Snippet: Oligonucleotide primers were obtained from Hokkaido System Science. pGEM-T (Promega), pGEX-4T3 (Amersham Bioscience), and pET32a (Novagen), respectively, were purchased. .. For PCR cloning, QUICK-Clone cDNA of mouse spleen and human HeLa cell (Clontech) were used as a template.

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: The PX domain cDNA (residues 12 to 135) generated by PCR was subcloned into pEGFP-N1 or pEYFP-N1 (Clontech, Mountain View, CA) to express PXK as a fusion to the N terminus of enhanced green fluorescent protein (EGFP) or enhanced yellow fluorescent protein (EYFP), respectively. .. The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden).

Article Title: Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Article Snippet: Vpr-related plasmids pLexA-VPR, pCMV-FLAG-VPR, pCDNA3-VPR, and pLexA-Tat were described previously ( ). pLexA-VPRL64A, VprL23A, VprL23,64,67A, and VprR80A were constructed by substituting the indicated amino acids of Vpr into the pLexA-VPR using a PCR-assisted in vitro mutagenesis reaction. pLexA-Vpr (64-96) was constructed by inserting the corresponding Vpr cDNA fragment into pLexA (Clontech, Palo Alto, Calif.). .. NL4-3GFPΔ Vpr was constructed by replacing the Nef coding sequence of the NL4-3GFPΔ Vpr obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, Md.) with the Nef-GFP fusion sequence of NL4-3GFP. pCDNA3-14-3-3η and pGEX-14-3-3η were constructed by introducing 14-3-3η cDNA into pCDNA3 (Invitrogen, Carlsbad, Calif.) and pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.), respectively. pB42AD-14-3-3η (1-244), (110-244), (141-244), (168-244), (190-210) and (210-244) were constructed by inserting 14-3-3η cDNA fragments of the indicated portions of 14-3-3η into pB42AD (Clontech). pGAD424-14-3-3σ (1-270) and (106-270) were constructed by subcloning 14-3-3σ cDNA fragments of 1-270 or 106-270 of 14-3-3σ into pGAD424 (Clontech), respectively.

Article Title: Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation
Article Snippet: .. GST pull down GST fusions were expressed from pGex-4T3 (Amersham) and untagged proteins were expressed from pRSF1b (Merck); in all cases full-length T. brucei RAD51 or RAD51 paralogue ORFs were cloned by PCR (primers available on request). .. Normally, Rosetta E. coli cells were transformed the GST fusion and untagged protein expression vectors to allow coexpression and GST pull down.

Positron Emission Tomography:

Article Title: Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus
Article Snippet: Plasmid constructions The pGEX-P and pET-N plasmids which contain sequences from the RSV Long strain have been described previously . .. For expression of GFP in E. coli , the EGFP gene was subcloned from pEGFP-N1 (Clontech) at the Sma I-Nco I sites in pGEX-4T3 (Amersham Biosciences).

Plasmid Preparation:

Article Title:
Article Snippet: The plasmid pHA-DYRK1A/S520A and the kinase-inactive mutants pHA-DYRK1A/K179R and pHA-DYRK1A/Y310,312F were generated by site-directed mutagenesis and verified by DNA sequencing. .. Plasmids expressing glutathione S -transferase (GST)-fusion proteins for wild-type DYRK1A (wt) and DYRK1A/K179R were generated by subcloning the fragments BamHI-SacI (amino acids 1–199) and SacI-NotI (amino acids 200–754) from their corresponding pHA-DYRK1A plasmids into the BamHI and NotI sites of pGEX-4T3 (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: For site directed mutagenesis, FLAG-Sprouty2 was subcloned into the Gateway vector system (Invitrogen) and point mutations were introduced using the QuickChange kit (Stratagene) according to manufacturer's protocol. .. FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ).

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences. .. The reporter constructs p5xGal4SV40-luc and p5xGal4tk-luc contain the luciferase gene and five copies of the GAL4 DNA binding sequence upstream of the simian virus 40 (SV40) promoter or the HSV thymidine kinase (TK) promoter and were described previously ( ).

Article Title: Sub-Nucleocapsid Nanoparticles: A Nasal Vaccine against Respiratory Syncytial Virus
Article Snippet: Paragraph title: Plasmid constructions ... For expression of GFP in E. coli , the EGFP gene was subcloned from pEGFP-N1 (Clontech) at the Sma I-Nco I sites in pGEX-4T3 (Amersham Biosciences).

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST. .. Plasmid pGEX-NMHC-IIA, encoding a fusion protein of GST and the rod domain of human NMHC-IIA, was described previously ( ) and used to generate GST-NMHC-IIA.

Article Title: GxcC connects Rap and Rac signaling during Dictyostelium development
Article Snippet: For expression in E . coli the digested N-terminal GxcC fragment was ligated into the BamHI site of pGEX 4T3 (GE Healthcare, N-terminal GST). .. This fragment was ligated in a pBluescript (Stratagene) vector and a BSR cassette was inserted in the Eco72I site of the resulting vector.

Article Title: The PRESAT-vector: Asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics
Article Snippet: T4 DNA ligase, Wizard plasmid miniprep kit, and Wizard SV DNA gel purification kit (Promega) were used for ligation and plasmid purification. .. Oligonucleotide primers were obtained from Hokkaido System Science. pGEM-T (Promega), pGEX-4T3 (Amersham Bioscience), and pET32a (Novagen), respectively, were purchased.

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: .. The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden). .. Human SNX27 was also cloned by PCR using the same cDNAs to amplify PXK and subcloned into pEGFP-N1 vector.

Negative Control:

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

shRNA:

Article Title: Nonmuscle Myosin Heavy Chain IIB Mediates Herpes Simplex Virus 1 Entry
Article Snippet: Plasmid pSSSP-Cre containing shRNA against Cre recombinase was described previously ( ). .. For generating a fusion protein of glutathione S -transferase (GST) and the rod domain of human NMHC-IIB (GST–NMHC-IIB), a plasmid (pGEX-NMHC-IIB) was constructed by amplifying the sequence containing NMHC-IIB codons 1672 to 1976 by PCR from pEGFP-BRF305 ( ) (a generous gift from M. Takahashi) and cloning the DNA fragment into pGEX-4T3 (GE Healthcare) in frame with GST.

In Vitro:

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ). .. In vitro transcribed and translated (IVTT) [35 S]-Methionine-labeled HA B-Raf was generated from pcDNA3 HA B-Raf using the TNT T7 Quick Coupled Transcription Translation kit (Promega) according to the manufacturer's protocol.

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Article Title: Vpr Protein of Human Immunodeficiency Virus Type 1 Binds to 14-3-3 Proteins and Facilitates Complex Formation with Cdc25C: Implications for Cell Cycle Arrest
Article Snippet: Vpr-related plasmids pLexA-VPR, pCMV-FLAG-VPR, pCDNA3-VPR, and pLexA-Tat were described previously ( ). pLexA-VPRL64A, VprL23A, VprL23,64,67A, and VprR80A were constructed by substituting the indicated amino acids of Vpr into the pLexA-VPR using a PCR-assisted in vitro mutagenesis reaction. pLexA-Vpr (64-96) was constructed by inserting the corresponding Vpr cDNA fragment into pLexA (Clontech, Palo Alto, Calif.). .. NL4-3GFPΔ Vpr was constructed by replacing the Nef coding sequence of the NL4-3GFPΔ Vpr obtained from the NIH AIDS Research and Reference Reagent Program (Rockville, Md.) with the Nef-GFP fusion sequence of NL4-3GFP. pCDNA3-14-3-3η and pGEX-14-3-3η were constructed by introducing 14-3-3η cDNA into pCDNA3 (Invitrogen, Carlsbad, Calif.) and pGEX-4T3 (Amersham Pharmacia Biotech, Piscataway, N.J.), respectively. pB42AD-14-3-3η (1-244), (110-244), (141-244), (168-244), (190-210) and (210-244) were constructed by inserting 14-3-3η cDNA fragments of the indicated portions of 14-3-3η into pB42AD (Clontech). pGAD424-14-3-3σ (1-270) and (106-270) were constructed by subcloning 14-3-3σ cDNA fragments of 1-270 or 106-270 of 14-3-3σ into pGAD424 (Clontech), respectively.

Selection:

Article Title: Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation
Article Snippet: GST pull down GST fusions were expressed from pGex-4T3 (Amersham) and untagged proteins were expressed from pRSF1b (Merck); in all cases full-length T. brucei RAD51 or RAD51 paralogue ORFs were cloned by PCR (primers available on request). .. In either case, a 50 ml culture of E. coli cells carrying the appropriate overexpression vectors, maintained by selection with antibiotics, were grown at 20°C until an OD of ∼ 0.5, at which time protein expression was induced with 1 mM IPTG overnight.

Produced:

Article Title: Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation
Article Snippet: GST-NIC (NIC1747-2444), GST-NICΔRAM (NIC1810-2444), GST-NICΔANK (NIC1747-1863 and 2077-2444), and GST-NICΔTAD (NIC1747-2193) were generated by subcloning NIC and its mutants into pGEX-4T3 (Amersham Pharmacia). .. His6 -XIAP, His6 -XIAPΔBIR1, His6 -XIAPΔBIR2, His6 -XIAPΔBIR3, and His6 -XIAPΔRF were produced by subcloning XIAP and its deletional mutants into pRSET-a (Invitrogen) for N-terminal His6 flanking.

Article Title: Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking ▿
Article Snippet: The bacterial vector expressing the PX domain of PXK was also generated by subcloning the corresponding cDNA into pGEX-4T3 (GE Healthcare, Uppsala, Sweden). .. All PXK mutants carrying one or two substitutions of amino acid residues were produced using QuikChange site-directed mutagenesis (Stratagene, La Jolla, CA).

Activation Assay:

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion
Article Snippet: The HA-Nup62, HA-Nup62-N (1 to 327), HA-Nup62-C (328 to 522), pVP16-Nup62, pVP16-Nup62-N (1 to 327), and pVP16-Nup62-C (328 to 522) genes were subcloned from the plasmid pAcGFPNup62WT ( ) into the pXJ41 expression vector using the HindIII and XhoI recognition sequences and into the pVP16-expressing vector (Clontech) downstream of the activation domain of the herpes simplex virus VP16 transactivator using the SalI and HindIII recognition sequences. .. The nsp1β gene and its mutant plasmids pM-K124A and pM-L126A were constructed by subcloning into the pM expression vector (Clontech) downstream of the yeast GAL4 DNA binding domain using the EcoRI and HindIII recognition sequences. pGEX-4T3-nsp1β and its mutant plasmids pGEX-4T3-K124A and pGEX-4T3-L126A were constructed by inserting nsp1β and its mutant K124A or L126A into the pGEX-4T3 (Amersham Pharmacia) expression vector using EcoRI and XhoI recognition sequences.

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: FLAG-mCLOCK, Myc-BMAL1, CMX-CLOCK, and CMX-MOP4 were kindly donated by Dr. P. Sassone-Corsi (University of California, Irvine, CA, USA) and Dr. Garret A. FitzGerald (University of Pennsylvania, Philadelphia, PA, USA; refs. , ). pVP16-CLOCK, which expresses CLOCK fused with the VP16 activation domain (AD), was constructed by subcloning the coding sequence of the mouse CLOCK into pVP16 (Clontech, Palo Alto, CA, USA) in an inframe fashion. .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega.

HAT Assay:

Article Title: Circadian rhythm transcription factor CLOCK regulates the transcriptional activity of the glucocorticoid receptor by acetylating its hinge region lysine cluster: potential physiological implications
Article Snippet: .. FLAG-CLOCK HAT Mut, which has replacements of its glycine, serine, and valine at amino acids 669, 670, and 672 with alanines, and thus, is defective in its HAT activity , was created by introducing the corresponding mutations into FLAG-CLOCK with a polymerase chain reaction (PCR)-assisted mutagenesis reaction. pRShGRα, pRSVerbA−1 , and pR107 were all gifts from Dr. R. M. Evans (Salk Institute, La Jolla, CA, USA) . pRShGRα expresses the human GRα, while pRSVerbA−1 contains the thyroid hormone receptor cDNA in an inverse orientation and was used as a negative control for pRShGRα. pR107 expresses the human GRα under the control of the SP6 promoter in vitro . pM-GRα full-length (FL) (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777), which express indicated portions of the human GRα fused with the GAL4 DBD, were constructed by subcloning the corresponding fragments of the human GRα cDNAs into pM (Clontech) in an inframe fashion. pGEX-4T3-GRα FL (amino acids 1–777), NTD (1–427), DBD (410–490), and LBD (485–777, which express the corresponding portions of the human GRα fused to the glutathione-S transferase (GST), were reported previously . pGEX-4T3-CLOCK FL (1–854), (1–370), and (371–854), which express the corresponding portions of mouse CLOCK fused with GST, were constructed by subcloning their cDNA fragments into pGEX-4T3 (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). pRShGRαK480A, K492A, K494/495A, K480/494/495A, K492/494/495A, and K480/492/494/495A were created by introducing the corresponding mutations that replace the indicated lysines with alanines using a PCR-assisted mutagenesis reaction with pRShGRα as a template. pMMTV-luc, which expresses luciferase under the control of the glucocorticoid-responsive MMTV promoter, was a gift of Dr. G. L. Hager (National Cancer Institute, Bethesda, MD, USA; ref. ). pRSV-RelA (p65) and pRSV-NF-κB I (p50), which, respectively, express p65 and p50 components of the nuclear factor-κB (NF-κB), were obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. (κB)3 -Luc, which expresses luciferase under the control of three κB-responsive elements (REs), was reported previously . pGAL4-E1B-TK-Luc, which expresses luciferase under the control of four GAL4-REs, was a gift from Dr. J. H. Segars (NIH, Bethesda, MD, USA; ref. ). pSG5 and pCMX, which are carrier plasmids for CLOCK, BMAL1, or MOP4, were purchased from Stratagene (La Jolla, CA, USA) and Promega (Madison, WI, USA), respectively, and were used as negative controls for these protein-expressing plasmids. pSV40-β-Gal was purchased from Promega. .. CLOCK, BMAL1, and control small-interfering RNAs (siRNAs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Recombinant:

Article Title: Sprouty2 association with B-Raf is regulated by phosphorylation and kinase conformation
Article Snippet: Recombinant human wild-type and V600E glutathione-S-transferase (GST)-B-Raf catalytic domains were from Cell Signaling Technology or Millipore. .. FLAG-Sprouty2 was cloned into pGEX-4T3 (GE Healthcare) and expressed and purified using standard methods ( ).

Article Title: Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation
Article Snippet: GST-NIC (NIC1747-2444), GST-NICΔRAM (NIC1810-2444), GST-NICΔANK (NIC1747-1863 and 2077-2444), and GST-NICΔTAD (NIC1747-2193) were generated by subcloning NIC and its mutants into pGEX-4T3 (Amersham Pharmacia). .. Recombinant GST fusion proteins were then purified on GSH agarose.

Article Title: Nox4 B-loop Creates an Interface between the Transmembrane and Dehydrogenase Domains *
Article Snippet: Paragraph title: Recombinant Protein Purification ... All PCR products were digested with BamHI and SalI and ligated into pGEX 4T3 (GE Healthcare) to generate glutathione S -transferase (GST) fusion proteins.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    GE Healthcare pgex 4t3 vector
    Preparation of H228A and D132A mutant proteins of CN2 with different N‐terminal additional sequences. cDNAs encoding inactive CN2 mutants, H228A, and D132A, were cloned into <t>pGEX‐6P3</t> and <t>pGEX‐4T3</t> vectors, respectively (Supporting Information Fig. S2), and expressed in E. coli as GST‐fusion proteins. The recombinant proteins were then trapped on glutathione‐Sepharose beads and digested with PreScission™ or thrombin protease, respectively. The yielded proteins have expected average molecular weights of 53,411 and 53,166 Da, respectively.
    Pgex 4t3 Vector, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgex 4t3 vector/product/GE Healthcare
    Average 99 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    pgex 4t3 vector - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    GE Healthcare pgex 4t3
    Preparation of H228A and D132A mutant proteins of CN2 with different N‐terminal additional sequences. cDNAs encoding inactive CN2 mutants, H228A, and D132A, were cloned into <t>pGEX‐6P3</t> and <t>pGEX‐4T3</t> vectors, respectively (Supporting Information Fig. S2), and expressed in E. coli as GST‐fusion proteins. The recombinant proteins were then trapped on glutathione‐Sepharose beads and digested with PreScission™ or thrombin protease, respectively. The yielded proteins have expected average molecular weights of 53,411 and 53,166 Da, respectively.
    Pgex 4t3, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgex 4t3/product/GE Healthcare
    Average 99 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    pgex 4t3 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    Image Search Results


    Preparation of H228A and D132A mutant proteins of CN2 with different N‐terminal additional sequences. cDNAs encoding inactive CN2 mutants, H228A, and D132A, were cloned into pGEX‐6P3 and pGEX‐4T3 vectors, respectively (Supporting Information Fig. S2), and expressed in E. coli as GST‐fusion proteins. The recombinant proteins were then trapped on glutathione‐Sepharose beads and digested with PreScission™ or thrombin protease, respectively. The yielded proteins have expected average molecular weights of 53,411 and 53,166 Da, respectively.

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Evidence for an essential role of intradimer interaction in catalytic function of carnosine dipeptidase II using electrospray‐ionization mass spectrometry

    doi: 10.1002/pro.2842

    Figure Lengend Snippet: Preparation of H228A and D132A mutant proteins of CN2 with different N‐terminal additional sequences. cDNAs encoding inactive CN2 mutants, H228A, and D132A, were cloned into pGEX‐6P3 and pGEX‐4T3 vectors, respectively (Supporting Information Fig. S2), and expressed in E. coli as GST‐fusion proteins. The recombinant proteins were then trapped on glutathione‐Sepharose beads and digested with PreScission™ or thrombin protease, respectively. The yielded proteins have expected average molecular weights of 53,411 and 53,166 Da, respectively.

    Article Snippet: The cDNA encoding D132A mutant protein was further subcloned into pGEX‐4T3 vector (GE Healthcare) using EcoRI and XhoI.

    Techniques: Mutagenesis, Clone Assay, Recombinant