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

Benjamin P C Chen, Tsonwin Hai

Research output: Contribution to journalArticle

70 Citations (Scopus)

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 [γ-32P]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.

Original languageEnglish (US)
Pages (from-to)73-75
Number of pages3
JournalGene
Volume139
Issue number1
DOIs
StatePublished - Feb 11 1994

Fingerprint

Escherichia coli Proteins
Histidine
Proteins
Myocardium
Phosphotransferases
Reading Frames
Nickel
Organism Cloning
Clone Cells
Adenosine Triphosphate
Phosphorylation
DNA
Genes

Keywords

  • heart muscle kinase
  • Histidine tag
  • nickel-chelating column
  • protein phosphorylation
  • T7 expression system

ASJC Scopus subject areas

  • Genetics

Cite this

Expression vectors for affinity purification and radiolabeling of proteins using Escherichia coli as host. / Chen, Benjamin P C; Hai, Tsonwin.

In: Gene, Vol. 139, No. 1, 11.02.1994, p. 73-75.

Research output: Contribution to journalArticle

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