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J Biomol Tech. 2009 Dec;20(5):241-8.

A pair of ligation-independent Escherichia coli expression vectors for rapid addition of a polyhistidine affinity tag to the N- or C-termini of recombinant proteins.

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  • 1Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield), Ontario, Canada.

Abstract

6x His tag is one of the most widely used affinity fusion tags that facilitates detection and purification of recombinant proteins. However, the location of this tag within a particular type of protein may influence the expression, solubility, and bioactivity of the protein, and the optimal location needs to be determined experimentally. To provide a tool for rapid generation of 6x His tags at the N- or C-terminus of any recombinant protein, we have constructed a pair of Escherichia coli expression vectors-pLIC-NHis and pLIC-CHis-based on the pET30a vector, for ligation-independent cloning (LIC). Construction of this new pair of LIC vectors was accomplished by replacement of the multiple cloning site of pET30a with two specifically designed LIC cloning sites. A target gene derived by PCR with a pair of predesigned primers can be inserted into the LIC site of pLIC-NHis for expression of recombinant proteins fused with the N-terminal sequence MHHHHHHG or into that of pLIC-CHis for expression of recombinant proteins with the C-terminal sequence THHHHHH. Successful expression of two normal mammalian prion proteins and five bacterial proteins in E. coli using this pair of LIC vectors reveals that these vectors are valuable tools for the production of recombinant His-tagged proteins in E. coli.

KEYWORDS:

His-tagged fusion protein; cloning; protein expression

PMID:
19949695
[PubMed - indexed for MEDLINE]
PMCID:
PMC2777343
Free PMC Article

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