Overexpression of monkey oviductal protein: purification and characterization of recombinant protein and its antibodies.
The secretory cells lining the lumen of the mammalian oviduct synthesize and secrete high molecular weight glycoprotein (OGP). Molecular cDNA cloning of most of the mammalian OGP has been accomplished. The nucleotide and deduced amino acid sequences show a remarkable homology across species and also to chitinase protein. Even though OGP has been shown to interact with gametes and the early embryo, the protein's direct function has not yet been established. A prerequisite for such studies is the availability of well-characterized protein in bulk. We used recombinant DNA technology to obtain OGP (rOGP). An authentic partial cDNA clone encoding bonnet monkey (Macaca radiata) OGP (accession number AF132 215) was recloned into expression vector pET20b. Overexpression of the protein could be demonstrated after induction with isopropylthio-beta-galactopyranoside. Recombinant protein was purified by gel filtration of Escherichia coli lysate through Sephadex G75. The protein migrated with a molecular weight of approximately 14 kDa on SDS-PAGE. The molecular weight as assessed by matrix-assisted laser adsorption time-of-flight was 14 439 daltons. With Western blot procedures the protein could be immunostained with antibodies to human OGP, baboon OGP, and antipeptide antibodies generated against a well-conserved region of mammalian OGP. The monospecificity of rabbit antibodies generated against rOGP was established by its ability to immunostain human OGP (100-110 kDa) isolated from hydrosalpinx by Western blot analysis, and the antibody immunostained epithelial cells that secrete OGP in human fallopian tubes. OGP binding sites on the head and tail region of monkey sperm could be demonstrated by using antibody against rOGP.
Natraj
Usha
U
Institute for Research in Reproduction, Indian Council of Medical Research, Parel, Mumbai 400012, India. dirirr@vsnl.com
Bhatt
Purvi
P
Vanage
Geeta
G
Moodbidri
S B
SB
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008255:Macaca radiata / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010215:Papio; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D013094:Spermatozoa / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D006023:Glycoproteins; C091468:OVGP1 protein, human; D011994:Recombinant Proteins