Overexpression and purification of tagged Escherichia coli proteins using a chromosomal knock-in strategy

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The purification of recombinant proteins from Escherichia coli (E. coli) has become a standard procedure both for research purposes and in biotechnology. One common way by which this is accomplished is by subcloning the gene of interest into a suitable expression vector and purifying the overexpressed protein using an affinity tag. In some cases, however, subcloning into plasmid vectors can be problematic. An alternative method could be to overexpress the gene of interest from the chromosome. Here, I describe a strategy to juxtapose strong transcriptional and translational sequences in front of any E. coli gene by recombination, which allows the gene product to be expressed in large quantities in the cell, and purified as a tagged protein. An application of this method to create a recombinant strain overexpressing the HrpA protein is described.

Original languageEnglish
Pages (from-to)294-298
Number of pages5
JournalProtein Expression and Purification
Volume46
Issue number2
DOIs
StatePublished - Apr 1 2006

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Escherichia coli Proteins
Purification
Genes
Escherichia coli
Proteins
Biotechnology
Chromosomes
Recombinant Proteins
Genetic Recombination
Plasmids
Research

Keywords

  • Protein expression
  • Protein purification
  • Recombination
  • Tagged proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

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abstract = "The purification of recombinant proteins from Escherichia coli (E. coli) has become a standard procedure both for research purposes and in biotechnology. One common way by which this is accomplished is by subcloning the gene of interest into a suitable expression vector and purifying the overexpressed protein using an affinity tag. In some cases, however, subcloning into plasmid vectors can be problematic. An alternative method could be to overexpress the gene of interest from the chromosome. Here, I describe a strategy to juxtapose strong transcriptional and translational sequences in front of any E. coli gene by recombination, which allows the gene product to be expressed in large quantities in the cell, and purified as a tagged protein. An application of this method to create a recombinant strain overexpressing the HrpA protein is described.",
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