摘要
根据GenBank中公布的粟酒裂殖酵母(Schizosaccharomyces pombe)N-糖酰胺酶(Pnglp)cDNA序列,设计并合成一对特异性引物,利用RT-PCR技术从粟酒裂殖酵母中克隆出糖酰胺酶cDNA。将得到的基因克隆到表达载体pET-15b中。重组质粒转入大肠杆菌BL21(DE3)中,经诱导表达和纯化提取后,进行酶活测定。实验结果表明,该酶的分子量约为39 kD,纯化后的重组N-糖酰胺酶可以对变性处理的糖蛋白进行糖链的切除,且这种作用需要还原剂DTT的辅助作用;N-糖酰胺酶只对错误折叠的糖蛋白有作用,对天然的糖蛋白没有作用。等量粟酒裂殖酵母Png1p在不同温度、pH、DTT浓度和底物变性温度下对等量核糖核酸酶B(RNase B)的脱糖基化检测发现,重组酶的最适反应温度30℃,最适反应pH为7.0,需要的最适DTT浓度为10 mmol/L,底物在100℃处理10 min时酶的脱糖基化率最高。
One pair of primers were designed and synthesized on the base of the cDNA sequence encoding Schizosaccharomyces pombe N-glycanase reported on the GenBank. The cDNA sequence encoding Peptide N-glycanase was cloned from the Schizosaccharomyces pombe by RT-PCR. And then the RT-PCR product was cloned into the expression vector pET-15b. The expression vector pET-15b（＋）/Pnglp was transformed into E. coli BL21（DE3）. The results showed that the relative molecular weight of the enzyme was determined to be approximately 39 kD using SDS-PAGE. The expression products after induction and purification can catalyze the cleavage of N-linked oligosaccharides from glycoprotein coped with heat, but have no action on the native glycoprotein with the help of DTT. The percentage of deglycosylated RNase B treated with equate Pnglp in different reaction temperature, pH, concentration of DTT and denatured temperature showed that the optimum temperature, the optimum pH is 30℃; the optimum concentration of DTT is 10 mmol/L and the optimum denatured temperature is 100℃.
出处
《生物工程学报》
CAS
CSCD
北大核心
2008年第4期592-597,共6页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目(No．30470049)资助~~
