人睾丸前列腺素D合成酶在毕赤氏酵母中的表达、纯化及鉴定

L PGDS是一种双功能蛋白 ,即催化PGD2产生和运输亲脂 疏水分子 .L PGDS主要分布于脑和男性生殖器官 ,并分泌到脑脊液、血清、精液、尿液等体液中 .测定体液中L PGDS的含量可辅助诊断一些神经系统疾病、生殖系统疾病、心血管疾病和肾脏疾病等 .在毕赤氏酵母中表达人睾丸前列腺素D合成酶 ,以利于进一步的生物学功能及临床应用研究 .用PCR的方法从质粒pGEX 2T htL PGDS上扩增出人睾丸L PGDS成熟肽基因编码序列 ,经测序证实后 ,将其插入到质粒pPIC9中 ,构建该基因的酵母表达质粒 .电转化毕赤氏酵母GS1 1 5 ,经甲醇诱导后 ,可实现L PGDS的高效、分泌性表达 .镍离子亲合层析法对表达上清进行纯化 ,SDS PAGE分析证实 ,在 2 7kD处有重组蛋白的表达 ,表达量为 2 7mg L .纯化蛋白可与视黄酸结合 ,使其紫外吸收波谱发生红移

Secretory Exprossion of Human Testis Prostaglandin D Synthase in Pichia pastoris

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.