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 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.
Dabrowski
S
S
Department of Microbiology, Technical University of Gdańsk, ul. Narutowicza 11/12, Gdańsk, 80-952, Poland.
Kur
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D006639:Histidine