Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host

Gene. 1994 Feb 11;139(1):73-5. doi: 10.1016/0378-1119(94)90525-8.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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

Substances

  • Oligodeoxyribonucleotides
  • Phosphorus Radioisotopes
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • Histidine
  • Adenosine Triphosphate
  • Protein Kinases
  • Endopeptidases

Associated data

  • GENBANK/L20317
  • GENBANK/L20318