Expression of Cucumber Ascorbate Oxidase gene in Microbes

黄瓜抗坏血酸氧化酶是一种分子大小约为130 kDa的二聚体糖蛋白。它是一种多铜酶，在催化中心有一个亚基含有4个铜原子。阐明铜酶的结构与功能之间的关系具有重要的意义。我们于1989年克隆并测序了黄瓜抗坏血酸氧化酶cDNA。同时，Huber及其同事建立了西葫芦抗坏血酸氧化酶的x射线晶体学三维结构分析。他们确定了铜原子的氨基酸配体。黄瓜和西葫芦抗坏血酸氧化酶的氨基酸序列同源性为89%。为了研究抗坏血酸氧化酶的结构和功能，位点定向和随机突变是一种有效的方法。由于黄瓜基因在微生物中的表达是此类研究的必要条件，我们尝试在大肠杆菌、短芽孢杆菌和酿酒酵母中克隆抗坏血酸氧化酶cDNA。虽然对微生物中更多的活性酶的合成没有成功，但给出了实验的总结。抗坏血酸氧化酶cDNA在微生物细胞中的表达。在tac启动子下，将抗坏血酸氧化酶cDNA连接到大肠杆菌高表达载体pKK233-2中，转化大肠杆菌JMIO5。IPTG诱导基因在log期表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和随后的免疫印迹分析显示形成了大量的抗坏血酸氧化酶蛋白。然而，大肠杆菌细胞在包涵体中积累蛋白质，在孵育温度、诱导时间、培养基成分、铜浓度等条件下均未检测到酶活性。尿素或胍类盐酸使包涵体变性，随后抗坏血酸氧化酶蛋白的重构不成功。短螺旋体分泌载体体系中未形成抗坏血酸氧化酶mRNA。在PH05启动子下经cDNA转化的酿酒酵母细胞合成的抗坏血酸氧化酶蛋白无酶活性。编码抗坏血酸氧化酶的基因组DNA的克隆。由于逆转录酶的误读，cDNA有可能具有不正确的核苷酸。以抗坏血酸氧化酶cDNA为探针，筛选黄瓜基因组文库，并通过基因行走法克隆出抗坏血酸氧化酶的全长基因组DNA。它包含4个外显子，编码区核苷酸序列与cDNA完全相同。酵母表达系统的修饰和烟草培养细胞的表达正在进行中。少

Cucumber ascorbate oxidase is a homodimer glycoprotein of molecular size of about 130 kDa. It is a muticopper enzyme and a subunit contains 4 copper atom in a catalytic center. It is important to elucidate the relation between the structure and the function of the copper enzyme. We have cloned and sequenced a cDNA for cucumber ascorbate oxidase in 1989. At the same time, Huber and coloeagues established the three-dimentional structure analysis of zucchini ascorbate oxidase by X-ray crystallography. They identified the amino acid ligands to Cu atom. The amino acid sequence homology between cucumber and zucchini ascorbate oxidase was 89%. To study, details of the structure and the function, site-directed and random mutations of ascorbate oxidase are a powerful approach. Since an expression of the cucumber gene in microbes is essential in such a study, we have tried recloning of the ascorbate oxidase cDNA in Escherichia coli, Bacillus brevis and Saccharomyces cerevisiae. Although the synt … More hesis of active enzyme in microbes did not succeed, summary of the experiment is shown.1. Expression of the ascorbate oxidase cDNA in microbial cells.The ascobate oxidase cDNA was ligated in an E. coli high expression vector, pKK233-2 under tac promoter, and transformed E. coli JMIO5. Gene expression was induced by IPTG in a log phase. Sodium laurylsulfate-polyacrylamide gel electrophoresis and the following Western blotting analysis revealed the formation of a large amount of ascorbate oxidase protein. However, E. coli cells accumulated the protein in an inclusion body and no enzyme activity was detected in several culture conditions including incubation temperature, induction time, medium component, copper concentration so on. Denaturation of the inclusion body by urea or guanidine hydrochloric acid, and the following reconstitution of the ascorbate oxidase protein was not successful. In B. brevis secretion vector system, no ascorbate oxidase mRNA was formed. Ascorbate oxidase protein synthesized in S. cerevisiae cells transformed by the cDNA under PH05 promoter showed no enzyme activity.2. Cloning of a genomic DNA encoding the ascornate oxidase.A cDNA has a possibility to have incorrect nucleotides by a miss-reading of reverse transcriptase. A genomic library of cucumber was screened by the ascorbate oxidase cDNA as a probe, and a full-length genomic DNA for the ascorbate oxidase was cloned by gene walking. It contained 4 exons and the nucleotide sequence in the coding region was completely same as that in cDNA. Modification of the expression system in yeast and expression in tobacco cultured cells are under going. Less