Refolding, structural transition and spermatozoa-binding of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli

An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca r adiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal sig nal sequence and the C-terminal transmembrane-like domain, was cloned in pQ E-30 vector and the protein expressed as inclusion bodies in Escherichia co li. Recombinant bmZPC (r-bmZPC) was solubilized from purified inclusion bod ies in the absence of a high concentration of chaotropic agents and was sub sequently refolded. Use of a low concentration of urea (2 M) during solubil ization of r-bmZPC helped to minimize the extent of protein aggregation dur ing refolding of the recombinant protein, and retain the existing native-li ke secondary structure that was essential for proper folding. Purified r-bm ZPC appeared as a dominant band of 43 kDa on SDS/PAGE and Western blot. Alt hough it lacked carbohydrate moieties, the purified and refolded r-bmZPC bo und to the head region of bonnet monkey spermatozoa, confirming the existen ce of a native-like conformation. CD revealed a maximum at 200 nm and a sin ,are broad minimum extending from 209 to 216 nm, indicating the presence of both alpha -helical and beta -sheet conformations in the refolded r-bmZPC. Two different phases of transition were observed by urea-gradient electrop horesis, suggesting the existence of multiple intermediate stages during th e unfolding of r-bmZPC. The availability of refolded r-bmZPC will help in e lucidating its role during the complex cascade of events during fertilizati on.