Abstract
The cleavage signal-1 protein (CS-1), a doublet antigen comprised of approx. 14-kDa and 18-kDa proteins has been shown to be present on the surface of sperm of various mammalian species including humans. Polyclonal antibodies to CS-1 inhibit the early cleavage of fertilized eggs without apparently affecting sperm penetration and pronuclear formation. We report here the cloning of the human CS-1 cDNA and its expression in vitro to obtain the recombinant protein (reCS-1) molecule. The CS-1 cDNA clone was isolated by immunological screening of a human testis λgt11 cDNA library with monospecific polyclonal antibody against CS-1. The cDNA is 1828 bp long; the start codon assigned to the first ATG (bp 98–100) encodes a protein with 249 amino acid residues terminating at TAA (bp 845–847). The cDNA isolated has a 97-bp 5′ and a 984-bp 3′ untranslated region. The potential polyadenylation signal (5′-AATAAA) is at bp 1803–1808. An extensive computer search of the GenBank database did not indicate any extensive homology with any known sequence, indicating that CS-1 is a unique protein. The CS-1 cDNA was cloned in the transcription vector, pGEM-11Zf, to obtain high-level in vitro transcription by SP6 and T7 RNA polymerase. The transcribed CS-1 RNA was translated in a rabbit reticulocyte in vitro translation system and produced a 33-kDa reCS-1 protein, as assessed by migration in a SDS-polyacrylamide gel. The polyclonal antibody against CS-1 specifically recognized the 33-kDa reCS-1 protein on Western blots of in vitro translated proteins, suggesting the authenticity of the cDNA clone. Besides enabling us to understand the molecular basis of signal transduction pathway(s) underlying fertilization and early cleavage, reCS-1 may find applications in the development of a contraceptive vaccine, and in the diagnosis and treatment of immuno-infertility in humans.
Published Version
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