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Cloning, Expression, Purification, and Immunocharacterization of Placental Protein-14

Dutta, Binita and Mukhopadhyay, Debaditya and Roy, Nita and Das, Goutam and Karande, Anjali A (1998) Cloning, Expression, Purification, and Immunocharacterization of Placental Protein-14. In: Protein Expression and Purification, 14 (3). pp. 327-334.

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Abstract

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.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to Academic Press.
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 11 Oct 2007
Last Modified: 19 Sep 2010 04:40
URI: http://eprints.iisc.ernet.in/id/eprint/12210

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