MOUSE ANDROGEN-DEPENDENT EPIDIDYMAL GLYCOPROTEIN CRISP-1 (DE AEG) - ISOLATION, BIOCHEMICAL-CHARACTERIZATION, AND EXPRESSION IN RECOMBINANT FORM/

In the rat, the secretory glycoprotein DE/AEG is one of the main const itutents of the epididymal fluid. We have recently reported the clonin g of the cDNA for the related cysteine-rich secretory protein-1 (CRISP -1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:1 92-198). The protein has now been isolated from the same organ and its N-terminal amino acid sequence has been determined. CRISP-1 exhibited an isoelectric point of similar to 6.8. High levels of CRISP-1 antige n were detected in the corpus and cauda of the epididymis, vas deferen s, seminal vesicle, prostate, and in the salivary gland by immunohisto chemistry. A quantitative analysis of the cauda epididymal fluid by sa ndwich ELISA revealed that CRISP-1 represented similar to 15% of the t otal protein. For heterologous expression, the CRISP-1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co-transfection of Sf9 cell s with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl malei mide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines local ized in the N-terminal moiety formed three bonds with each other, sugg esting the existence of two discrete domains in the protein. (C) 1995 Wiley-Liss, Inc.