DOG ZONA-PELLUCIDA GLYCOPROTEIN-3 (ZP3) - EXPRESSION IN ESCHERICHIA-COLI AND IMMUNOLOGICAL CHARACTERIZATION

An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and th e C-terminal transmembrane-like domain, was amplified by polymerase ch ain reaction from a full-length cDNA clone. The amplified SacI and Pst I restricted fragment was cloned in-frame downstream of the T5 promote r under lac operator control for expression in the pQE-30 vector. Reco mbinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protei n in Escherichia coli. Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-beta-D-thiogalactopyronoside. Immunoblots with a muri ne monoclonal antibody, MA-451 (raised against porcine ZP3 beta-a homo logue of DZP3 and crossreactive with dog zona pellucida), revealed a m ajor band of 42 kDa. Localization studies revealed that the recombinan t protein was present only in an insoluble intracellular fraction. Fur ther optimization studies revealed that the level of expression of rec -DZP3 was significantly higher in Luria broth medium containing glycer ol rather than glucose and maximum expression was observed when cultur es were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compar ed to 4 mg/L from a shake flask culture. Immunization of two male rabb its with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP 3 conjugated to diphtheria toroid generated high antibody titers again st rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune se rum reacted with porcine ZP3 beta but failed to react with porcine ZP3 alpha in a Western blot, Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive f luorescence with zona pellucida, The availability of rec-DZP3 will hel p in evaluating its efficacy for fertility regulation in stray dogs, ( C) 1998 Academic Press.