链霉菌UDP-葡萄糖脱氢酸编码基因Ste6的克隆表达和性质研究

链霉菌139能够产生一种全新的胞外多糖——依博素（139A）,该多糖体内具有显著抗类风湿性关节炎活性。其生物合成基因簇（GenBank Accession Number：AYl31229）已被鉴定约31．3kb,包含22个开放阅读框（ste1—ste22）。以pET-30a为载体,克隆并在大肠杆菌BL21（DE3）中进行了ste6基因的表达,对该基因的克隆、表达与性质进行了研究。亲和层析法证实,纯化后重组蛋白具有催化UDP-葡萄糖脱氢变成UDP-葡萄糖醛酸的活性。这表明ste6编码产物是葡萄糖脱氢酶。为了证实ste6基因与依博素生物合成的关系,采用单交换基因破坏策略构建了ste6基因阻断突变株。结果初步显示ste6和依博素生物合成相关。

Cloning,Expression and Characterization of the Ste6 Gene Encoding a UDP-glucose Dehydrogenase in Streptomyces

Streptomyces sp.139 was identified to produce a new exopolysaccharide Ebosin(139A) with anti-rheumatic arthritis activity in vivo.The Ebosin biosynthesis gene cluster(31.3 kb; GenBank Accession Number :AY131229)containing 22 ORFs (ste1-ste22) of Streptomyces sp.139 had been reported previously.In this paper,we present experimental evidence for the identity of the ste6 gene product as a UDP-glucose dehydro genase (UDPGDH).With pET-30a as vector,the gene was cloned and expressed in E scherichia coli BL21(DE3).The expressed protein was purified to homogeneity by His-Bind resin affinity chromatograpy and it was able to catalyze UDP-glucose to UDP-glucuronic acid.To evaluate the function of ste6,t he gene was disrupted by a single-crossover homologous recombination event and the result showed that ste6 is required in Ebosin biosynthe sis.