Abstract

To establish an effective approach for inducing expression of the RecQ core of Bloom’s Syndrome protein (BLM^642-1290) and assaying its biological activity in vitro, BLM^642-1290 recombinant protein was expressed with IPTG at room temperature inEscherichia coli, and then the expressed product was assayed using SDS-PAGE and western blotting. After purification via affinity chromatography, DNA binding activity and unwinding activity of the protein were assessed by fluorescence polarization. Furthermore, the ATPase activity of the protein was also assayed using ultraviolet spectrophotometry based on PiColorLock Gold reagent. An effective expression method was established for BLM protein in E. coli. The obvious bioactivities of the protein were observed in binding to ssDNA or dsDNA, unwinding the dsDNA in the presence of ATP, as well as catalyzing ATP hydrolysis in the presence of ssDNA in vitro. The prokaryocyte expression method of BLM^642-1290was established successfully and the protein with biological activity was obtained from recombinant E.coli. This would be significant to provide a better understanding on BLM protein and facilitate the elucidation of mechanism of pathopoiesia in Bloom’s Syndrome.

Key words: BLM^642-1290 protein, induced expression, enzymatic activity.

Abbreviation

Abbreviations: BS, Bloom’s Syndrome; BLM, Bloom Syndrome protein; BLM^642-1290, the RecQ core of Bloom’s Syndrome protein; SCEs, sister-chromatid exchange; HRDC, helicase RNase D conserved domain; IPTG, isopropyl-1-thio-α-D-galactopyranoside; RecQ-Ct, RecQ Conserved -Terminal region; BSA, bovine serum albumin; PMSF, phenylmethanesulfonyl fluoride; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; ATP, adenosine triphosphate; SDS-PAGE,sodium dodecyl sulfate-polyacrylamide gel electrophoresis.