Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host.
We have constructed two convenient vectors to produce foreign proteins in Escherichia coli. The first vector was developed to produce histidine (His)-tagged fusion proteins. In addition to encoding six contiguous His residues, it contains three unique restriction sites that allow cloning of a blunt-ended DNA fragment in three different reading frames. Therefore, one can clone any gene of interest in this vector to make a fusion protein tagged with six His at the N terminus. The His-tag allows purification of the fusion protein to almost homogeneity by a nickel-chelating column in a single step. The second vector is a derivative of the first vector; it encodes two tandem phosphorylation sites for heart muscle kinase (HMK) immediately downstream from the His residues. Therefore, the resulting fusion protein can be radiolabeled using [gamma-32P]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.
Chen
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BP
Ohio State Biochemistry Program, Ohio State University, Columbus 43210.
Hai
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D010450:Endopeptidases; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D006639:Histidine; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000096:biosynthesis* / Q000302:isolation & purification; D011865:Radioisotope Dilution Technique; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping; D013728:Terminator Regions, Genetic
D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010450:Endopeptidases