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 constituents 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 approximately 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 approximately 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.
Eberspaecher
U
U
Research Laboratories of Schering AG, Berlin, Germany.
Roosterman
D
D
Krätzschmar
J
J
Haendler
B
B
Habenicht
U F
UF
Becker
A
A
Quensel
C
C
Petri
T
T
Schleuning
W D
WD
Donner
P
P
D000595:Amino Acid Sequence; D000728:Androgens / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004822:Epididymis / Q000737:chemistry*; D005260:Female; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008562:Membrane Glycoproteins; D051379:Mice; D008969:Molecular Sequence Data; D011467:Prostate / Q000737:chemistry; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012469:Salivary Glands / Q000737:chemistry; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014649:Vas Deferens / Q000737:chemistry
D000728:Androgens; C486321:Crisp1 protein, mouse; C486320:Crisp3 protein, rat; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides