Secretory  Exprossion  of  Human Testis Prostaglandin D Synthase in Pichia pastoris

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Chinese Journal of Biochemistry and Molecular Biology ›› 2003, Vol. 19 ›› Issue (06) : 757-762.

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Secretory Exprossion of Human Testis Prostaglandin D Synthase in Pichia pastoris

GAO Yun, HUANG Yu feng , XIA Xin yi, MA Bai kun

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Lipocalin type prostaglandin D synthase(L\|PGDS) is bifunctional, acting as a PGD2 producing enzyme and as a potential lipophilic ligand transporter. L PGDS is localized mainly in the central nervous system and male genital organs of various mammls and is secreted into the cerebrospinal fluid, serum, seminal plasma and urine. Its concentration in these body fluids is useful for diagnosis of neurological disorders, dysfunction of sperm formation,and cardiovascular and renal diseases. To express Human prostaglandin D synthase in Pichia pastoris for further research on biological function and clilnical applications. Human testis L PGDS gene coding region was amplified from plasmid pGEX 2T/htL PGDS by PCR with a deletion of the signal peptide sequence. The DNA fragment was inserted into pPIC9 to construct yeast expression plasmid followed by transformation of the yeast GS115 strain with electroporation. The recombinant protein was induced to express by methnol and purified with Ni 2+ NTA resin. The recombinat protein was found with a molecular mass of 27 kD on SDS PAGE and an expression level as high as 27 mg/L. After forming the complex formation with purified L PGDS, the UV spectra of all trans retinoic acid were red shifed approximately 30 nm.

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prostaglandin D synthase / yeast expression system / metal chelation chromatography / glycosylation

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GAO Yun, HUANG Yu feng , XIA Xin yi, MA Bai kun. Secretory Exprossion of Human Testis Prostaglandin D Synthase in Pichia pastoris[J]. Chinese Journal of Biochemistry and Molecular Biology, 2003, 19(06): 757-762

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Received	Revised	Published
2003-12-20	2003-12-20	2003-12-20
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2003-12-20

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