Reference - Detail
|Author||Chen BP, Hai T.|
|Title||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.
|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|
|WOS Category||GENETICS & HEREDITY|
|Prokaryotes E. coli||pETHis pETHisK|