Dog Zona Pellucida Glycoprotein-3 (ZP3): Expression inEscherichia coliand Immunological Characterization

An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplifiedSacI andPstI restricted fragment was cloned in-frame downstream of the T5 promoter underlacoperator control for expression in the pQE-30 vector. Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein inEscherichia coli.Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-β-d-thiogalactopyronoside. Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β-a homologue of DZP3 and cross-reactive with dog zona pellucida), revealed a major band of 42 kDa. Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction. Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture. Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with porcine ZP3β but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with zona pellucida. The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs.