Expression of human proacrosin in Escherichia coli and finding to zona pellucida

Proacrosin is a multifunctional protein present in the sperm acrosome. This study characterizes the expression of human proacrosin in bacteria and ass esses zona pellucida binding activity. The cDNA encoding human proacrosin w as subcloned in pGEX-3X and pET-22b vectors. In the pGEX system, expression of the full-length fusion protein was not detected. In the pET system, an expression product with an apparent molecular size similar to that expected for the proenzyme (Rec-40, 42-44 kDa) was recognized by a monoclonal antib ody to human acrosin, AcrC5F10. A 32-34-kDa protein (Rec-SO), not recognize d by AcrC5F10 on Western blots, was the major expression product. Proteins of 21 (Rec-20) and 18 (Rec-10) kDa were recovered as insoluble expression p roducts as were Rec-40 and Rec-30, and truncated products from the C termin us were detected in the soluble fraction. Rec-40 and Rec-30 coexisted at an y culture time tested. Immune serum raised against Rec-30 (AntiRec-30) stai ned the acrosomal region of permeabilized human spermatozoa and recognized the recombinant proteins and proacrosin from human sperm extracts. Amino ac id sequence analysis indicated that Rec-30, Rec-20, and Rec-10 are N-termin al fragments of proacrosin. The recombinant proteins Rec-40, -30, -20, and -10 were found to interact with homologous I-121-zona pellucida glycoprotei ns.