Purified and refolded recombinant bonnet monkey (macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa

Govind, Chhabi K. ; Gahlay, Gagandeep K. ; Choudhury, Sangeeta ; Gupta, Satish K. (2001) Purified and refolded recombinant bonnet monkey (macaca radiata) zona pellucida glycoprotein-B expressed in Escherichia coli binds to spermatozoa Biology of Reproduction, 64 (4). pp. 1147-1152. ISSN 0006-3363

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Official URL: http://www.biolreprod.org/content/64/4/1147.full

Related URL: http://dx.doi.org/10.1095/biolreprod64.4.1147

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 a helical and β 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.

Item Type:Article
Source:Copyright of this article belongs to Society for the Study of Reproduction.
Keywords:Fertilization; Ovum; Sperm
ID Code:12040
Deposited On:16 Nov 2010 13:57
Last Modified:16 May 2016 21:26

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