Reference - Detail
| 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 |
| DOI | 10.1016/0378-1119(94)90525-8 |
| 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 | 2.984 |
| Times Cited | 65 |
| WOS Category | GENETICS & HEREDITY |
| Resource | |
| Prokaryotes E. coli | pETHis pETHisK |