中国生物工程杂志

Abstract Phospholipase C gamma-1(PLC-γ1) is a nerve-enriched protein containing SH2-SH2-SH3 domain. It has been demonstrated that the SH2 domain of PLC-γ1 regulates the phosphorylation of PLC -γ1 through interaction with receptor tyrosin kinase , and the SH3 domain of which interacts with sosorvinculinto mediate Ras signaling and regulate actin polymerization. To understand better this potential functional role and produce the monoclonal antibody against SH2-SH2-SH3 of PLC-γ1，using RT-PCR from adult human cortex ,the functional domain（SH2-SH2-SH3 domain）ofPLC-γ1 was cloned to pGEX-2T fusion vector and transformed into E.coli BL21 host cells. SDS-PAGEand Western blot analysis revealed that the induced recombinant protein by IPTG was expressed successfully at 33℃ but weakly at 37℃. Furthermore, the recombinant protein was degraded at 37℃ . The GST fusion protein extract was immobilised on glutathione sepharose 4B bead and eluted with glutathion. Taken together, the results indicated that a lower culture batch(33℃) was utilized to produce recombinant protein easily. The fragment expression of SH2-SH2-SH3 at higher temperature(37℃), however, suggests that PLC-γ1 degradation was involved not only in eukaryotic cell but also in prokarytic cell(BL21 strain). The identification of cleaved site of SH2-SH2-SH3 domain by peptide sequencing analysis will be done in the future