根毛農桿菌十異戊二烯雙磷酸 合成脢基因之選殖與表現

Abstract

According to the known sequence of decaprenyl diphosphate synthase ( dps ) gene in Agrobacterium tumerfaciens, we designed the cloning primers for PCR cloning of this gene from Agrobacterium rhizogenes strain 1610. The total length of dpsAg1610 is 1,077 bp. Comparing to A. tumerfaciens, the dpsAg1610 gene has 92.4 % and 98.4 % similarity in DNA and amino acid sequence. The alignment shows there are two conserved sequence DDXXD, which is regarded as catalytic domain, in dpsAg1610. After transformation into Escherichia coli, which produces CoQ8 predominantly, the expression was induced by adding IPTG. In SDS-PAGE and Western blot analysis, we confirmed that rDPS-Ec was successfully expressed in E. coli. The molecular weight of rDPS-Ec is 41 kDa, which is the same as prediction. And the best expression level of recombinant protein was achieved by 0.1mM IPTG induction for 20 hours. CoQ content was analyzed by reverse phase HPLC with photo diode detection. We hardly can observed the production of CoQ10. Furthermore, we tried to constitutively express dpsAg1610 in Pichia pastoris. However, neither rDPS was expressed, nor CoQ10 was detected in Pichia transformants. In the future work, we need to improve our extraction efficiency and the HPLC conditions. And try to get over the problem in Pichia system for rDPS expression.