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

摘要: An internal cDNA fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (bmZPC), excluding the N-terminal signal sequence and C-terminal transmembrane-like domain, was cloned in pQE-30 vector protein expressed as inclusion bodies Escherichia coli. Recombinant bmZPC (r-bmZPC) solubilized from purified absence of a high concentration chaotropic agents subsequently refolded. Use low urea (2 m) during solubilization r-bmZPC helped minimize extent aggregation refolding recombinant protein, retain existing native-like secondary structure that essential for proper folding. Purified appeared dominant band 43 kDa on SDS/PAGE Western blot. Although it lacked carbohydrate moieties, refolded bound head region spermatozoa, confirming existence conformation. CD revealed maximum at 200 nm single broad minimum extending 209 216 nm, indicating presence both α-helical β-sheet conformations r-bmZPC. Two different phases transition were observed by urea-gradient electrophoresis, suggesting multiple intermediate stages unfolding The availability will help elucidating its role complex cascade events fertilization.