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

Polymerase chain reaction (PCR)-derived DNA fragment containing the complet e structural gene for SSB protein of the Escherichia coli was cloned into a n expression vector. The clones expressing His-Lagged SSB protein were sele cted. The cloned DNA fragments were verified to be authentic by sequencing several clones. The recombinant SSB protein (His-tagged SSB) contained a po lyhistidine tag at the N-terminus (38 additional amino acids) that allowed single-step isolation by Ni2+ affinity chromatography, We found that recomb inant plasmids are unstable and give a low level of expression in E. coli B L21(DE3) strain. However, the plasmids were stable in E. coli BL21(DE3) con taining the pLysS plasmid, which suppresses expression prior to induction, and His-tagged proteins were highly expressed upon IPTG addition. The SSB p rotein 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-t agged SSB protein to increase amplification efficiency with a number of div erse templates. The use of SSB protein may prove to be generally applicable in improving PCR efficiency, (C) 1999 Academic Press.