磷脂酰肌醇特异性磷脂酶C的异源表达和应用

doi:10.13560/j.cnki.biotech.bull.1985.2019-0630

摘要/Abstract

摘要： 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OD_600nm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1 322.5 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。

关键词: 磷脂酰肌醇特异性磷脂酶C, 蜡样芽孢杆菌, 密码子优化, 酶切

Abstract: 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.

Key words: phosphatidylinositol-specific phospholipase C, Bacillus cereus, codon optimization, enzyme digestion

林美璇, 周小满, 关锋, 崔文璟. 磷脂酰肌醇特异性磷脂酶C的异源表达和应用[J]. 生物技术通报, 2020, 36(1): 81-87.

LIN Mei-xuan, ZHOU Xiao-man, GUAN Feng, CUI Wen-jing. Heterologous Expression and Application of Phosphatidylinositol-specific Phospholipase C[J]. Biotechnology Bulletin, 2020, 36(1): 81-87.

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