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Anal Biochem. 2012 Oct 15;429(2):132-9. doi: 10.1016/j.ab.2012.07.016. Epub 2012 Jul 22.

A strategy for the expression of recombinant proteins traditionally hard to purify.

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1
Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

Abstract

We have developed a series of plasmid vectors for the soluble expression and subsequent purification of recombinant proteins that have historically proven to be extremely difficult to purify from Escherichia coli. Instead of dramatically overproducing the target protein, it is expressed at a low basal level that facilitates the correct folding of the recombinant protein and increases its solubility. Highly active recombinant proteins that are traditionally difficult to purify are readily purified using standard affinity tags and conventional chromatography. To demonstrate the utility of these vectors, we have expressed and purified full-length human DNA polymerases η, ι, and ν from E. coli and show that the purified DNA polymerases are catalytically active in vitro.

PMID:
22828411
PMCID:
PMC3575598
DOI:
10.1016/j.ab.2012.07.016
[Indexed for MEDLINE]
Free PMC Article
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