Identification of the 48-kDa G11 protein from guinea pig testes as sperad

A protein found specifically in the membrane of spermatozoa called G11 has been implicated in sperm–egg binding and fusion. This study describes purification and identification of the G11 antigen. The G11 protein was purified using anion exchange chromatography, immunoaffinity chromatography and preparative SDS‐PAGE and was subjected to amino acid microsequencing by tandem mass spectrometry. Internal amino acid sequence data derived from the 48‐kDa G11 protein revealed that G11 is the recently discovered guinea pig sperm protein, sperad. Sperad is a transmembrane protein present in the periacrosomal plasma membrane of guinea pig sperm. The cytoplasmic domain of sperad was amplified from a guinea pig testes cDNA expression library by polymerase chain reaction and cloned into a prokaryotic gene expression vector, pGEX‐2T. The recombinant glutathione S‐transferase fusion protein was immunoblotted with monoclonal antibody G11. The results obtained from this study confirmed the monoclonal antibody G11 epitope location on the cytoplasmic domain of sperad. J. Exp. Zool. 293:617–623, 2002. © 2002 Wiley‐Liss, Inc.