Penicillin-binding protein 2a of Streptococcus pneumoniae: Expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cel l wall. PBPs possess transpeptidase and transglycosylase activities respons ible for the final steps of the bacterial cell wall crosslinking and polyme rization, respectively, To facilitate our structural studies of PBPs, we co nstructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-t erminal part of the protein including the transmembrane domain) of the pbp2 a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli. This GST fusion form of PBP2a , designated GST-PBP2a*, was expressed almost exclusively as inclusion bodi es. Using a combination of high- and low-speed centrifugation, large amount s of purified inclusion bodies were obtained. These purified inclusion bodi es were refolded into a soluble and enzymatically active enzyme using a sin gle-step refolding method consisting of solubilization of the inclusion bod ies with urea and direct dialysis of the solubilized preparations. Using th ese purification and refolding methods, approximately 37 mg of soluble GST- PBP2a* protein was obtained from 1 liter of culture. The identity of this r efolded PBP2a* protein was confirmed by N-terminal sequencing. The refolded PBP2a*, with or without the GST-tag, was found to bind to BOCILLIN FL, a b eta-lactam, and to hydrolyze S2d, an analog of the bacterial cell wall stem peptides. The S2d hydrolysis activity of PBP2a* was inhibited by penicilli n Gr. In conclusion, using this expression system, and the purification and refolding methods, large amounts of the soluble GST-PBP2a* protein were ob tained and shown to be enzymatically active. (C) 1999 Academic Press.