Cloning,Heterologous Expressionand Application Of Phosphatidylinositol-Specific Phospholipase C From Bacillus Cereus

Phosphatidylinositol-specific phospholipase C（PI-PLC）is a phospholipase C that can act on the phosphodiester bond of phosphatidylinositol（PI,PIP,PIP2）.PI-PLC not only has broad prospects in industrial applications,but also is a research tool in the laboratories,and has broad potential value as a substitute to antibiotics to resist parasites.The study of PI-PLC purification and its enzymic activity properties is one of the hotspots in many laboratories around the world.PI-PLC is widely expressed in many microorganisms.It is reported that PI-PLC derived from Bacillus cereus does not act as a virulence factor.Therefore,PI-PLC derived from Bacillus cereus is selected for heterologous expression in E.coli.For better expression,PI-PLC codons were optimized according to the preference of E.coli codons.The gene expression vectors pET-28a-PI-PLC and pGEX-6P-1-PI-PLC were constructed and transformed into E.coli BL21/DE3,respectively.The results showed that the recombinant PI-PLC GST-tag could be purified well by expressing PI-PLC using vector pGEX-6P-1-PI-PLC.The optimal induction conditions were as follows:5%of inoculum size,the OD₆₀₀ at 0.5 and24 h of induced culture with 0.3 mmol·L^-1 IPTG at 16°C.After expression and purification under optimal induction conditions,the obtained PI-PLC was used in the following studies.After preliminary exploration of the enzymatic properties,it was found that:1)the specific enzyme activity of purified recombinant PI-PLC was 1322.5 U·mg^-1;2)the ability of recombinant PI-PLC to hydrolyze the standard substrate reached the highest at 75°C;3)recombinant PI-PLC showed relative effective activity at high pH;4)Co²⁺and Ca²⁺ions significantly promoted the enzyme activity of recombinant PI-PLC,and Mg²⁺ions slightly inhibited the enzyme activity of recombinant PI-PLC;5)recombinant PI-PLC was stable when stored at 4°C.Treatment of adherent cells using recombinant PI-PLC in PBS buffer at 37°C revealed that recombinant PI-PLC had a good digestion effect on GPI-anchored protein CD59 on the surface of human invasive bladder cancer cells YTS-1,non-invasive bladder cancer cells KK47,and bladder transitional cell carcinoma cells J82.PI-PLC treatment for 1.5 h and longer could achieve a better digestion effect,and cell density was negatively correlated with the digestion effect.After fusion and expression of GPI signal peptide and Neu-1 gene,one of the sialidases,Neu-1 protein which was intracellularly expressed can be expressed on the cell membrane of YTS-1 cells with GPI anchor structure.This translocation from the intracellular to the cell membrane in constructed cells can be simply determined by in situ digestion with recombinant PI-PLC obtained in this experiment.In summary,the heterologous expression and purification of PI-PLC were achieved in this study.The recombinant PI-PLC showed high enzymatic activity and could effectively cleave GPI-anchored proteins on cell surface,which could be applied to the study of GPI-anchored proteins in the future.