PURIFICATION AND MOLECULAR-CLONING OF CARP OVARIAN CYSTATIN

The ovarian fluid of carp consists of many components. Using the antis erum against carp serum, Western blot analysis of ovarian fluid was do ne in order to distinguish substances synthesized by the ovary from th ose derived from the serum. Several ovary-specific substances were det ected including a protein of 12 kDa (p12), which was purified to homog eneity. Purified p12 displays a single band in SDS-PAGE under nonreduc ing condition and it can inhibit the enzymatic activity of papain with an apparent inhibition constant of 0.01 nM. The primary structure of p12 was partially determined by Edman degradation and fully elucidated by molecular cloning. A cDNA of 531 bp encoding p12 was obtained. The precursor of p12 has 129 residues, including a signal peptide of 18 r esidues and a mature protein of 111 residues. The N- and C-terminus of p12 are threonine and methionine, respectively. The p12 shares many c ommon features of the family 2 cystatins of other species, including t he similarity of the protein size (in the range of 110 to 120 residues ), the presence of 4 cysteine residues and the occurrence of invariant residues throughout the molecule.