肠膜明串珠菌蔗糖磷酸化酶基因在大肠杆菌中的表达

Expression of Sucrose Phosphorylase Gene from Leuconostoc mesenteroides in Escherichia coli

以肠膜明串珠菌基因组DNA为模板,通过PCR扩增得到1 581 bp的蔗糖磷酸化酶(SPase)DNA片段。将该基因克隆到表达载体pET-22b(+)上,构建获得重组质粒pET-SPase。测序结果与GenBank上已公布的基因序列比较,有1个碱基发生变化,但该碱基的改变未引起氨基酸序列的改变。将pET-SPase转化到Escherichia coli Rosetta(DE3)感受态中,IPTG诱导表达后进行SDS-PAGE分析,目的蛋白条带约为55 kD,与预期大小一致,结果表明SPase基因在大肠杆菌中进行了表达。酶活分析,产物的比活为1.8 U/mg,证明了表达产物具有预期的酶活性。进一步考察了IPTG浓度、诱导温度和时间等因素对重组菌表达的蔗糖磷酸化酶的影响。在优化条件下,该蔗糖磷酸化酶的比活可以达到16.6 U/mg,比优化表达条件前的酶比活提高了9.2倍,比已报道的肠膜明串珠菌粗酶液比活(7.1 U/mg)提高了2.34倍。

The Spase-encoding gene was amplified from Leuconostoc mesenteroides genome DNA using PCR technique,and the PCR product was approximately 1 581 bp DNA segment.Plasmid sequencing showed that one base was different from the sequence announced in GenBank but did not change the amino acid sequence.The constructed recombinant plasmid was transformed to Escherichia coli Rosetta（DE3）by heat shock method and expressed under induction of IPTG.The result showed that the cloned SPase with molecular weight 55 kD and specific enzyme activity 1.8 U/mg was expressed in Rosetta.The influences of induction conditions such as IPTG concentration,induction temperature and time of induction on the expression of the recombinant protein were investigated.Under optimal condition,the specific enzyme activity could reach 16.6 U/mg,which is 9.2 times higher than that of primary expression condition,and 2.34 times higher than that of sucrose phosphorylase（7.1 U/mg）that has been reported already.