title: Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host.
abstract: 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.
authors.affiliation: Ohio State Biochemistry Program, Ohio State University, Columbus 43210.
authors.forename: B P
authors.initials: BP
authors.lastname: Chen
authors.forename: T
authors.initials: T
authors.lastname: Hai
mesh_terms: 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
chemical_list: D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006639:Histidine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010450:Endopeptidases