Heterologous Expression and Application of Phosphatidylinositol-specific Phospholipase C

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

Abstract

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

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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