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Purified and Refolded Recombinant Bonnet Monkey (Macaca radiata) Zona Pellucida Glycoprotein-B Expressed in Escherichia coli Binds to Spermatozoa¹

^a Gamete Antigen Laboratory, National Institute of Immunology, New Delhi 110 067, India

ABSTRACT

Bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (bmZPB), excluding the N-terminal signal sequence and the C-terminus transmembrane-like domain, has been expressed in Escherichia coli as polyhistidine fusion protein. A requirement of 4 M urea to maintain the purified protein in soluble state rendered it unsuitable for biological studies. Purification of refolded r-bmZPB without urea and devoid of lower molecular weight fragments was achieved by following an alternate methodology that involved purification of inclusion bodies to homogeneity and solubilization in the presence of a low concentration of chaotropic agent (2 M urea) and high pH (pH 12). The solubilized protein was refolded in the presence of oxidized and reduced glutathione. The circular dichroism spectra revealed the presence of both helical and ß sheet components in the secondary structure of the refolded r-bmZPB. The binding of the refolded r-bmZPB to the spermatozoa was evaluated by an indirect immunofluorescence assay and also by direct binding of the biotinylated r-bmZPB. The binding was restricted to the principal segment of the acrosomal cap of capacitated bonnet monkey spermatozoa. In the acrosome-reacted spermatozoa a shift in the binding pattern of r-bmZPB was observed and it bound to the equatorial segment, postacrosomal domain, and midpiece region. Binding of biotinylated r-bmZPB was inhibited by cold r-bmZPB as well as by monoclonal and polyclonal antibodies generated against r-bmZPB. These results suggest that nonglycosylated bmZPB binds to capacitated as well as acrosome-reacted spermatozoa in a nonhuman primate and may have a functional role during fertilization.

FOOTNOTES

First decision: 25 September 2000.

¹ The contribution by C.K.G. and G.K.G. may be considered equal. This work was supported by grants from the Department of Biotechnology and Ministry of Health and Family Welfare, Government of India. Support for a part of this work (CSA-98-219/CSA-99–262) was also provided by the Contraceptive Research and Development (CONRAD) Program, Eastern Virginia Medical School, Norfolk, Virginia. G.K.G. is a recipient of the research fellowship of Council of Scientific and Industrial Research, Government of India. The views expressed by the authors do not necessarily reflect the views of the funding agencies.

² Correspondence: Satish K. Gupta, Staff Scientist-VI and Chief, Gamete Antigen Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India. FAX: 91 11 6162125; skgupta{at}nii.res.in

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