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.
Ilayperuma
Isurani
I
Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, Dunedin New Zealand. isurani@anato.ruh.ac.lk
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000737:chemistry; D013737:Testis / Q000737:chemistry*
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins