Soluble expression of pbp3 of Streptococcus pneumoniae in Escherichia coli and identification of its activity

Abstract:

[Objective] To study the structure and application of penicillin-binding protein 3 (PBP3), the pbp3 gene of Streptococcus pneumoniae R6 was cloned and the recombinant protein of PBP3 was expressed in Escherichia coli with expression vector pGEX-6p-pbp3*. [Methods] The truncated pbp3 gene (15?413 aa) was cloned from S. pneumoniae R6 with PCR, and the pbp3* fragment digested by BamH Ⅰand Xho Ⅰwas inserted into pGEX-6p-1 to generate pGEX-6p-pbp3* expression plasmid. GST-PBP3* fusion protein was expressed in cytoplasm of E. coli BL21(DE3) and purified with Glutathione-Sepharose 4B column. The recombinant fusion protein, GST-PBP3, was firstly digested by PreScission Protease for cutting off the GST tag, and then pass through the Glutathione-Sepharose 4B column for getting the purified PBP3 protein. The PBP3 activity was identified by its binding activity to β-lactam antibiotics, such as cefquinome. [Results] The truncated pbp3 gene was verified by sequence analysis and the pGEX-6p-pbp3* expression plasmid was constructed successfully. Finally the soluble PBP3 protein with biologic activity was purified. [Conclusion] The active recombinant PBP3 was successfully obtained from prokaryotic expression system, and this laid a firm foundation for further study of the structure and application of PBP3.

Key words:Streptococcus pneumoniae, PBP3, soluble expression, purification, Binding activity