关键词: 酿酒酵母；无机焦磷酸酶；大肠杆菌

Abstract:

Objective To express Saccharomyces cerevisiae(S.cerevisiae) inorganic pyrophosphatase (Y-IPPA) inEscherichia coli (E.coli) and obtain the active yeast inorganic pyrophosphatase. Methods S. cerevisiae  genome was used as a template,the yeast inorganic pyrophosphatase sequences were obtained by searching NCBI, the primers were designed,the yeast inorganic pyrophosphatase gene sequence was obtained by PCR.It was inserted in the T-simple 19 cloning vector and transformed into amplification of E.coli DH5α,the recombinant plasmid was se
quenced and verified correctly,the restriction fragments were inserted into prokaryotic expression vector pET28 (a),and then transformed into E.coli DH5α and then transformed into E.coli BL21 (DE3) after restriction analysis and sequencing correctly. Y-IPPA was expressed by IPTG induction.The protein purification system was used to purify the proteins and the protein level, activity and specific activity were determined. Results The T-simple 19 consistent with the Y-IPPA base sequence was obtained and the recombinant expression vector pET28 (a)-Y-IPPA was constructed,and transformed into E. coli BL21 (DE3) with IPTG induction and SDS-PAG Eelectrophoresis. The molecular weight of 32 000 was noted in Y-IPPA protein bands.Nickel affinity chromatography was performed to obtain the target protein.The protein level, activity and specific activity  were 0.312 g.L^-1,31.13 units.mL^-1,and 99.73uints.mg^-1.Conclusion The Y-IPPA protein is obtained successfully,and it lays the foundation for further study on its enzymatic features.

Key words:  saccharomyces cerevisiae;inorganic pyrophosphatase;Escherichia coli