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

被引:94
作者
EBERSPAECHER, U
ROOSTERMAN, D
KRATZSCHMAR, J
HAENDLER, B
HABENICHT, UF
BECKER, A
QUENSEL, C
PETRI, T
SCHLEUNING, WD
DONNER, P
机构
[1] Research Laboratories of Schering Ag, Berlin
关键词
EPIDIDYMIS; GLYCOPROTEIN; SPERM; SALIVA;
D O I
10.1002/mrd.1080420205
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In the rat, the secretory glycoprotein DE/AEG is one of the main constitutents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine-rich secretory protein-1 (CRISP-1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192-198). 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 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP-1 represented similar to 15% of the total 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 cells with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N-terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein. (C) 1995 Wiley-Liss, Inc.
引用
收藏
页码:157 / 172
页数:16
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