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130 kDa的同源二聚体糖蛋白。它是一种多铜酶，其催化中心含有4个铜原子。阐明铜酶的结构和功能之间的关系是非常重要的。1989年，我们克隆并测序了黄瓜抗坏血酸氧化酶的一个基因。同时，Huber等人建立了西葫芦抗坏血酸氧化酶的X射线结晶学三维结构分析方法。他们将氨基酸配基确定为铜原子。黄瓜与西葫芦抗坏血酸氧化酶的氨基酸序列同源性为%。为了研究抗坏血酸氧化酶的结构和功能的细节，定点突变和随机突变是一种强有力的方法。由于黄瓜基因在微生物中的表达在这样的研究中是必不可少的，我们尝试在大肠杆菌、短杆菌和酿酒酵母中重新克隆抗坏血酸氧化酶基因。虽然SYNT…更多的微生物中的活性酶没有成功，实验总结如下。抗坏血酸氧化酶基因在微生物细胞中的表达：将抗坏血酸氧化酶基因连接到含有tac启动子的大肠杆菌高效表达载体pKK233-2中，转化大肠杆菌JMIO5。IPTG在对数生长期诱导基因表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和随后的Western blotting分析显示有大量抗坏血酸氧化酶蛋白的形成。然而，在培养温度、诱导时间、培养基组分、铜浓度等培养条件下，大肠杆菌细胞以包涵体形式积累蛋白质，没有检测到酶的活性。用尿素或盐酸胍对包涵体进行变性，随后的抗坏血酸氧化酶蛋白重组不成功。在短小芽孢杆菌分泌载体系统中，未形成抗坏血酸氧化酶基因。结论：1.在PH05启动子下转化的酿酒酵母细胞中合成的抗坏血酸氧化酶蛋白无酶活性。编码抗坏血酸氧化物酶的基因组DNA的克隆。由于逆转录酶的误读，cdna有可能含有不正确的核苷酸。以抗坏血酸氧化酶基因为探针，筛选黄瓜基因组文库，用基因步行法克隆了黄瓜抗坏血酸氧化酶全长基因组DNA。该基因由4个外显子组成，编码区核苷酸序列与cDNA完全一致。酵母表达系统的改造和烟草培养细胞的表达正在进行中。较少

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