Protein purification, sequencing, and cDNA cloning of P68: A sperm surface protein

P68/62, an {dollar}sim{dollar}68 kDa doublet, is a sperm surface protein whose ability to bind to the egg zona pellucida has been documented in vitro and in vivo (Tanphaichitr et al., 1992, 1993). This study aimed to purify P68/62 from boar sperm for P68 protein microsequencing and to isolate the cDNA for P68 from boar testis. Early attempts were made to isolate the P68 cDNA from boar testis using reverse-transcription PCR and primers based on previously obtained P68 peptide sequences. One PCR product, clone A, showed 82% and 78% overall nucleotide and amino acid homology to human cleavage signal 1 (HCS1) protein, respectively. Clone A is most likely the pig homolog of HCS1. No other clones sequenced showed any potential as the P68 cDNA clone. Therefore, more P68/62 was purified to obtain more protein sequences for future P68 cDNA cloning work. P68/62 was isolated from a boar sperm protein extract using chromatofocusing in conjunction with fast pressure liquid chromatography. Pure P68/62 fractions were defined by silver-stained SDS-PAGE and immunoblotting results using antiSLIP1, antiRSA, and anti-keratin. Approximately 0.5 {dollar}mu{dollar}g of pure P68 was obtained for microsequencing. Trypsin digestion of P68 and sequencing of four peptides resulted in sequences highly homologous to a casein peptide and three different regions of the human testis arylsulfatase-A protein. Preliminary analyses and studies suggest that AS-A and the casein peptide are components of the P68 polypeptide.