磷脂酰肌醇特异性磷脂酶C的异源表达和应用

旨在研究大肠杆菌产磷脂酰肌醇特异性磷脂酶C(PI-PLC)的发酵表达和分离纯化,探究PI-PLC酶切GPI锚定蛋白的效果。依据NCBI数据库中蜡样芽孢杆菌的PI-PLC的基因序列,按照大肠杆菌的密码子偏好性进行密码子优化,合成相应基因序列并构建基因表达载体pGEX-6P-1-PI-PLC。将重组质粒转入受体菌E.coli BL21(DE3)中,通过加入异丙基硫代-β-D-半乳糖苷(IPTG)诱导目的基因PI-PLC表达。经检测,含有GST标签的PI-PLC融合蛋白以可溶蛋白形式存在于菌体破碎上清,分子量约为61 kDa,与预期相符。初步优化诱导表达条件后,发现最佳诱导表达条件为:以接种量5%接种体积分数接种,待菌体生长至OD600nm达到0.5,在16℃条件下以0.3 mmol/L浓度IPTG诱导24 h。利用GST标签对蛋白进行纯化,纯化后的PI-PLC质量浓度为0.52 mg/mL,比酶活为1322.5 U/mg。利用PI-PLC酶液对哺乳动物细胞表面的模式GPI锚定蛋白CD59进行酶切,酶切作用显著。因此,下一步可以将PI-PLC融合蛋白应用于细胞生物学中对GPI-APs的研究和鉴定。

Heterologous Expression and Application of Phosphatidylinositolspecific Phospholipase C

This work aims to optimize the fermentation conditions and purify the phosphatidylinositol-specific phospholipase C(PI-PLC)exogenously expressed in Escherichia coli,and also to evaluate the enzymolysis effect of PI-PLC on glycosylphosphatidylinositol-anchored protein(GPI-AP).In this study,we synthesized the codon-optimized gene sequence of PI-PLC from Bacillus cereus in the NCBI database,according to the codon preference of E.coli,and inserted the sequence into plasmid pGEX-6P-1.The recombinant plasmid pGEX-6P-1-PI-PLC was transformed into E.coli BL21(DE3),and expression of PI-PLC was induced by isopropylthio-β-D-galactoside(IPTG).SDS-PAGE analysis revealed that the GST-tagged PI-PLC fusion protein of was presented as a soluble protein in the supernatant of cell lysate,its molecule weight was about 61 kD,which was consistent with the predicted.The optimized induction conditions for PI-PLC expression were obtained as follows:5%of inoculum size,induced 24 h with 0.3 mmol/L IPTG at 16℃when the OD 600nm reached 0.5.The protein was further purified with GST purification kit,the concentration of purified PI-PLC was 0.52 mg/mL,and the specific enzyme activity was 1322.5 U/mg.The PI-PLC enzymatic solution significantly digested the model GPI-anchored protein CD59 on the surface of mammal cells.Therefore,PI-PLC obtained in this study can be used to study and identify the GPI-Aps in cytobiology in the future.