Functional expression of RubisCO by a high-speed carbon dioxide fixing microorganism

嗜热嗜热假单胞菌TH-1菌株具有Rubisco基因下的cbbQ基因(CbbLS)。CbbQ蛋白被认为参与了RuBisCO的翻译后激活/稳定。我们发现在cbbQ基因下存在cbbO基因，并对该基因进行了测序。下一步，构建了含有cbbQ、cbbO、cbbQ-cbbO基因的重组表达载体。用其中一种表达载体和含有cbbLS基因的表达载体对大肠杆菌进行转化。然后，分别通过测定Rubisco活性或通过凝胶电泳法分析Rubisco活性或表达的差异。当只使用含有cbbLS的重组质粒时，Rubisco活性较低，在聚丙烯酰胺凝胶电泳法上检测到Rubisco蛋白为一条拖尾带。相反，在cbbQ或cbbO基因的存在下，Rubisco的活性增加了2~3倍，并且在聚丙烯酰胺凝胶电泳中检测到了一条清晰的条带。在单独的实验中，制备了三种蛋白质(CbbLS、CbbQ和CbbO)，用于体外激活Rubisco。在我们的研究过程中，CbbO蛋白本身被证明具有CO_2结合活性，这将是未来研究的一个思路来源。

A thermophilic hydrogen bacterium Pseudomonas hydrogenothermophila strain TH-1 possesses cbbQ gene under RubisCO gene (cbbLS). CbbQ protein was thought to participate in the post-translational activation/stabilization of RubisCO.We showed the presence of cbbO gene under cbbQ gene and clarified the gene sequence. In the next step, plasmids harboring cbbQ,cbbO,cbbQ-cbbO gene were prepared. By using one of the above plasmids and a plasmid harboring cbbLS,transformation of E.coli was performed. Then, difference in activity or expression of RubisCO was analyzed by measuring RubisCO activity or by electrophoreis, respectively. When only the plasmid harboring cbbLS was used, RubisCO activity was low and the RubisCO protein was detected as a smear band on polyacrylamide-gel electrophoresis. Contrary to this, in the presence of cbbQ or cbbO gene, the activity of RubisCO increased 2 to 3 fold, also the RubisCO protein was detected as a clear band on polyacrylamide-gel electrophoresis.In a separate experiments, three kinds of protein (CbbLS,CbbQ,and CbbO) were prepared for the experiment of in vitro RubisCO activation. However, successful results have not been obtained.During our research, CbbO protein itself was proved to possess CO_2 incorporation activity, which will be a source of thinking for future research.