RRC ID 33382
Author Chen BP, Hai T.
Title Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host.
Journal Gene
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.
Volume 139(1)
Pages 73-5
Published 1994-2-11
PII 0378-1119(94)90525-8
PMID 8112591
MeSH Adenosine Triphosphate / metabolism Amino Acid Sequence Animals Base Sequence Blotting, Western Chromatography, Affinity / methods Cloning, Molecular / methods Endopeptidases Escherichia coli / metabolism* Genetic Vectors* Histidine Humans Molecular Sequence Data Myocardium / enzymology Oligodeoxyribonucleotides Phosphorus Radioisotopes Phosphorylation Plasmids Promoter Regions, Genetic Protein Kinases / biosynthesis* Protein Kinases / isolation & purification Radioisotope Dilution Technique Reading Frames Recombinant Fusion Proteins / biosynthesis* Recombinant Fusion Proteins / isolation & purification Recombinant Proteins / biosynthesis* Recombinant Proteins / isolation & purification Restriction Mapping Terminator Regions, Genetic
IF 2.638
Times Cited 62
Prokaryotes E. coli pETHis pETHisK