A T7 expression vector for producing N- and C-terminal fusion proteins with glutathione S-transferase.
The pGEX system for protein production in E. coli is widely used in molecular biology. A bacterial expression vector, pETGEXCT, which incorporates features of the pGEX and pET expression systems was designed. pETGEXCT allows the production of N- and C-terminal fusions to glutathione S-transferase (GST) under the tight control of the T7 promoter. Use of this vector can circumvent problems associated with unstable or inactive fusions to the N terminus of GST. Indeed, it is demonstrated that fusions to the N terminus of the eukaryotic DNA-binding protein, RSRFC4, cannot be tolerated. Fusion of RSRFC4 to the N terminus of GST in the pETGEXCT vector is a prerequisite to purify the RSRFC4 DNA-binding domain in an active form using glutathione-agarose affinity chromatography.
Sharrocks
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Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, UK.
D000595:Amino Acid Sequence; D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D016384:Consensus Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis*; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; D008969:Molecular Sequence Data; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping; D012549:Schistosoma japonicum / Q000201:enzymology / Q000235:genetics*; D013917:Thrombin / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D004268:DNA-Binding Proteins; D026161:MADS Domain Proteins; D064326:MEF2 Transcription Factors; C578035:MEF2A protein, human; D018006:Myogenic Regulatory Factors; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase; D013917:Thrombin