title: Production of tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid and dog zona pellucida glycoprotein-3 for contraceptive vaccine development.
abstract: Affinity tags can interfere in various physicochemical properties and immunogenicity of the recombinant proteins. In the present study, tag-free recombinant fusion protein encompassing promiscuous T cell epitope of tetanus toxoid [TT; amino acid (aa) residues 830-844] followed by dilysine linker and dog zona pellucida glycoprotein-3 (ZP3; aa residues 23-348) (TT-KK-ZP3) was expressed in Escherichia coli. The recombinant protein, expressed as inclusion bodies (IBs), was purified by isolation of IBs, processed to remove host cell proteins, followed by solubilization and refolding. A specific 39 kDa protein including ZP3 was identified by SDS-PAGE. CD spectra showed the presence of α-helices and β-sheets, and fluorescent spectroscopy revealed emission maxima of 265 A.U. at 339 nm for refolded protein and showed red shift in the presence of 6 M guanidine hydrochloride. Immunization of inbred FvB/J female mice with purified recombinant TT-KK-ZP3 (25 μg/animal) led to generation of high antibody titers against the recombinant protein. The antibodies reacted specifically with ZP matrix surrounding mouse oocytes. Immunized mice showed significant reduction in fertility as compared to the control group. The studies described herein provide a simple method to produce and purify tag-free recombinant protein for the development of a contraceptive vaccine.
authors.affiliation: Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Gupta
authors.forename: Abhinav
authors.initials: A
authors.lastname: Shrestha
authors.forename: Amulya Kumar
authors.initials: AK
authors.lastname: Panda
authors.forename: Satish Kumar
authors.initials: SK
authors.lastname: Gupta
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004285:Dogs; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005260:Female; D002479:Inclusion Bodies / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013745:Tetanus Toxoid / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D022642:Vaccines, Contraceptive / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins
chemical_list: D004527:Egg Proteins; D018984:Epitopes, T-Lymphocyte; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D013745:Tetanus Toxoid; D022642:Vaccines, Contraceptive; D000072216:Zona Pellucida Glycoproteins; C000606521:Zp3 protein, mouse