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Cloning, overexpression, and purification of the recombinant His-tagged SSB protein of Escherichia coli and use in polymerase chain reaction amplification.

Authors
  • Dabrowski, S
  • Kur, J
Type
Published Article
Journal
Protein expression and purification
Publication Date
Jun 01, 1999
Volume
16
Issue
1
Pages
96–102
Identifiers
PMID: 10336866
Source
Medline
License
Unknown

Abstract

Polymerase chain reaction (PCR)-derived DNA fragment containing the complete structural gene for SSB protein of the Escherichia coli was cloned into an expression vector. The clones expressing His-tagged SSB protein were selected. The cloned DNA fragments were verified to be authentic by sequencing several clones. The recombinant SSB protein (His-tagged SSB) contained a polyhistidine tag at the N-terminus (38 additional amino acids) that allowed single-step isolation by Ni2+ affinity chromatography. We found that recombinant plasmids are unstable and give a low level of expression in E. coli BL21(DE3) strain. However, the plasmids were stable in E. coli BL21(DE3) containing the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were highly expressed upon IPTG addition. The SSB protein was purified by metal-affinity chromatography on Ni2+-TED-Sepharose columns. The enzyme was characterized by fluorescence titration experiments for single-stranded DNA binding activity. We have applied the use of His-tagged SSB protein to increase amplification efficiency with a number of diverse templates. The use of SSB protein may prove to be generally applicable in improving PCR efficiency.

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