PROTEIN EXPRESSION AND PURIFICATION 12, 331–339 (1998) ARTICLE NO. PT970869 Dog Zona Pellucida Glycoprotein-3 (ZP3): Expression in Escherichia coli and Immunological Characterization Ramasamy Santhanam, Amulya K. Panda,* V. Senthil Kumar, and Satish K. Gupta 1 Gamete Antigen Laboratory and *Product Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India Received August 18, 1997, and in revised form December 8, 1997 Key Words: recombinant zona pellucida glycopro- tein-3; intracellular expression; inclusion bodies; fer- An internal fragment (978 bp) corresponding to the mentation; immunogenicity; immunofluorescence. dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by poly- merase chain reaction from a full-length cDNA clone. Mammalian oocyte is enveloped by a unique, acellu- The amplified SacI and PstI restricted fragment was lar, translucent matrix termed zona pellucida (ZP 2 ). cloned in-frame downstream of the T5 promoter under lac operator control for expression in the pQE-30 vec- The ZP mediates the initial recognition and binding tor. Recombinant DZP3 (rec-DZP3) was expressed as a events of sperm to the oocyte in a relatively species- polyhistidine fusion protein in Escherichia coli. Opti- specific manner, thus playing a vital role in fertiliza- mum expression of rec-DZP3 was observed at 1.0 mM tion. This critical role during fertilization has made isopropyl - b - D - thiogalactopyronoside. Immunoblots the ZP glycoproteins potential candidate antigens for with a murine monoclonal antibody, MA-451 (raised immunocontraception. In most species, ZP is composed against porcine ZP3b-a homologue of DZP3 and cross- of three biochemically and immunologically distinct reactive with dog zona pellucida), revealed a major glycoproteins: ZP1, ZP2, and ZP3 (1). Considerable evi- band of 42 kDa. Localization studies revealed that the dence suggests that in mouse, hamster, and human, recombinant protein was present only in an insoluble ZP3 serves as the putative primary sperm receptor (2 – intracellular fraction. Further optimization studies re- 4). Expression of ZP3 in mice and marmosets is re- vealed that the level of expression of rec-DZP3 was stricted to growing oocytes (5, 6). However, recent ex- significantly higher in Luria broth medium containing perimental evidences suggest that in cynomolgous glycerol rather than glucose and maximum expression monkeys and humans, ZP3 may also be synthesized by was observed when cultures were induced during the granulosa cells (7, 8). mid-log phase of growth. Batch fermentation with The cDNA sequence for ZP3 from mouse, hamster, glycerol as the carbon source yielded 30 mg/L of rec- dog, porcine, marmoset, bonnet monkey, and human DZP3 compared to 4 mg/L from a shake flask culture. has revealed 67–94% interspecies homology (1, 3, 6, 9, Immunization of two male rabbits with Ni-NTA-puri- 10). Immunological cross-reactivity of ZP glycoproteins fied rec-DZP3 and two female dogs with the rec-DZP3 among various species, due to sequence homology, has conjugated to diphtheria toxoid generated high anti- led to the possibility of heterologous immunization (11). body titers against rec-DZP3 as determined by en- The availability of large numbers of pig ovaries from zyme-linked immunosorbent assay. Rabbit immune se- rum reacted with porcine ZP3b but failed to react with abattoirs has made pig ZP glycoproteins promising can- porcine ZP3a in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with 2 Abbreviations used: A, absorbance; aa, amino acid(s); bp, base pairs; IPTG, isopropyl-b-D-thiogalactopyronoside; kDa, kilodalton; zona pellucida. The availability of rec-DZP3 will help Ni-NTA, nickel – nitrilotriacetic acid; nt, nucleotide(s); PCR, polymer- in evaluating its efficacy for fertility regulation in ase chain reaction; rec, recombinant; ZP, zona pellucida; DZP3, dog stray dogs.  1998 Academic Press zona pellucida-3; DT, diphtheria toxoid; CFA, complete Freund’s ad- juvant; IFA, incomplete Freund’s adjuvant; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; HRPO, 1 To whom correspondence should be addressed. Fax: 91-11- 6162125. E-mail: GA@ nii.ernet.in. horseradish peroxidase; SIZP, solubilized isolated zona pellucida. 331 1046-5928/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.