胱硫醚β-合酶的原核表达、纯化及其活性研究-生物化学与分子生物学专业论文.docxVIP

华中科技 大学硕士 学 华 中 科 技 大 学 硕 士 学 位 论 文 II II Abstract The two sulfur-containing amino acids, methionine and cysteine, are metabolically linked via the transsulfuration pathway. In mammals, methionine is an essential amino acid, which can be converted via homocysteine to cysteine by the reverse transsulfuration pathway. The first reaction in this two-step sequence is catalyzed by cystathionine β-synthase, which condenses homocysteine and serine to give cystathionine. This is followed by a γ-elimination catalyzed by cystathione γ-lyase, which cleaves cystathionine to cysteine and a-ketoglutarate. Recent researches showed that homocysteine is an independent risk factor for erosclerosis in hyperlipidemic patients. In normal, The CBS-catalyzed condensation of serine and homocysteine generates cystathionine for the removal of sulfur from methionine and homocysteine when the levels of these amino acids are elevated. Cystathionine β-synthase (CBS) deficiency is the most common cause of homocystinuria. The recombinant expression plasmid pGEX-4T-1-CBS was transformed into E.coli BL21 (DE3). The induce condition were optimized at 25 ℃, 12 h, and the expressed fusion protein CBS-6his is soluble. The fusion protein was purified by Sepharose-NI affinity column and G-25 column. The molecular mass of the fraction cleaved by thrombin determined by SDSwas about 63 kDa. So the CBS-6his fusion protein was successfully expressed in E. coli. The CBS-catalyzed condensation of serine and homocysteine generates cystathionine which was detected by the HPLC. It shows that the CBS-6his fusion protein is active. Key words： cystathionine β-synthase, recombinant plasmid, Prokaryotic Expression, protein purification, HPLC PAGE IV PAGE IV 目 录 摘 要 I HYPERLINK \l _bookmark0 Abstract II HYPERLINK \l _bookmark1 目 录 III HYPERLINK \l _bookmark2 1 绪论 1 HYPERLINK \l _bookmark3 1.1 胱硫醚 Β-合酶的研究进展 1 HYPERLINK \l _bookmark4 1.2 原核表达技术 5 HYPERLINK \l _bookmark5 1.3 高效液相色谱 6 HYPERLINK \l _bookmark6 1