可溶性泛素样特异性蛋白酶1的表达及活性鉴定

摘要

Obective:To obtain souble expressive ubiquitin-like specific protease 1(ULP1).Methods:ULP1 gene was optimized according to the preference codon usage of E.coli.ULP1 gene was synthesized and then cloned into pGEX-6P-1 vector,and transformed into E.coli BL21(DE3) competent cells.Inducing time and concentration of IPTG were optimized,and ULP1 was induced by 0.5 mmol/L IPTG at 37℃ for 5 h.Results:The best inducing conditions of was in 0.1 mmol/L IPTG,and expressed as a souble fusion protein.The catalytic reaction of ULP1 to SUMO-GFP showed high specificity and activity.Conclusion:The souble fusion ULP1 with biological activity was successful expressed.%目的:高效可溶性表达泛素样特异性蛋白酶1(ULP1).方法:根据大肠杆菌密码子的偏好性优化合成编码ULP1的基因片段序列,将其克隆到原核表达载体pGEX-6P-1中,转化大肠杆菌BL21(DE3),用0.5 mmol/L IPTG于37℃诱导表达8h,观察重组蛋白ULP1的表达情况;优化诱导时间及IPTG浓度,并鉴定重组蛋白ULP1的生物学活性.结果:重组蛋白ULP1表达的最佳条件为37℃、0.1 mmol/L的IPTG诱导表达5h,目的蛋白以可溶性表达为主;Western印迹结果表明,重组蛋白ULP1能够被His单克隆抗体识别,重组蛋白ULP1能够特异性酶切SUMO-GFP.结论:表达了具有生物学活性的SUMO蛋白酶ULP1.