Cloning, expression, purification, and immunocharacterization of placental protein-14.
Human placental protein-14 (PP-14), a member of the lipocalin superfamily, shares homology at the level of the primary and secondary structures with bovine beta-lactoglobulin. It is the most prominent endometrial protein synthesized by the glandular cells of endometrium under estrogen priming and progesterone stimulation. The temporal and spatial expression of PP-14 in the female reproductive tract combined with its biological activities ex vivo suggest that this glycoprotein probably plays an essential physiological role in the regulation of fertilization, implantation, and maintenance of pregnancy. We proposed to elucidate the molecular mechanisms involved in the function of this protein. A prerequisite to such investigations on any protein is the availability of sufficient amounts of the same in a homogenous form. Therefore, recombinant DNA technology was employed. The PP-14 cDNA was obtained from the first-trimester endometrial tissue RNA by RT-PCR using unique primers. After confirming the identity of the gene, the protein was expressed in Escherichia coli and purified to homogeneity. The gene was also cloned and expressed in Pichia pastoris to obtain the protein product in a glycosylated form. The recombinant proteins were immunocharacterized using a cross-reactive antibody raised to bovine beta-lactoglobulin. Polyclonal antiserum raised to the E coli expressed PP-14 also bound to the native PP-14 from amniotic fluid suggesting that recombinant PP-14 may be exploited to elucidate functional aspects of the protein.
Dutta
B
B
Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012.
Mukhopadhyay
D
D
Roy
N
N
Das
G
G
Karande
A A
AA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D003429:Cross Reactions; D004717:Endometrium / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005822:Genetic Vectors / Q000235:genetics; D000071816:Glycodelin; D006023:Glycoproteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007106:Immune Sera; D007782:Lactoglobulins / Q000276:immunology; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D011261:Pregnancy Trimester, First; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000071816:Glycodelin; D006023:Glycoproteins; D007106:Immune Sera; D007782:Lactoglobulins; C045246:PAEP protein, human; D011257:Pregnancy Proteins; D011993:Recombinant Fusion Proteins