Expression、Purification And Crystallization Of Malonyl-Coenzy A Reductase-a Key Enzyme Of The3-Hydroxypropionate Cycle

A novel carbon dioxide fixation pathway,3-hydroxypropionate (3-HP) cycle, has been discovered in thermophilic filamentous anoxygenic photosynthetic (FAP) bacteria. Roseiflexus castenholzii, the experimental object in this paper, is one of the newly found FAP species. The characteristic of this pathway is 3-HP as a metabolic intermediate, which can be used for synthesis of many valuable products in chemical industry. In addition, there are several bifuctionional enzymes in this pathway, such as the malony-CoA reductase (MCR), which catalyzes the conversion of malony-CoA to 3-HP. Since MCR is not involved in any other known metabolic pathways, it can be regarded as a characteristic enzyme of the 3-HP cycle. The study on structure and function of MCR not only can help us know the detail of 3-HP formation process, but also can uncover the catalytic mechanism of these bifuctionional enzymes. The improvements in the understanding of 3-HP cycle are beneficial to the development of production biologically.In this project, we have utilized molecular biology and protein chemistry technology to study the MCR and carried out the following experiments:1) Based on the bioinformatics analysis of genome of Roseiflexus castenholzii in JGI databank, the specific primers were designed to clone the mcr gene by PCR. The PCR products were ligated to a new type of expression plasmid pEASY-E1, and then the recombinant vector was transformed into the expression host cell strain E.coli BL21(DE3).2) By using the methods of affinity chromatography, ion exchange chromatography and gel filtration chromatography, the heterogeneous expression of MCR protein was isolated and the purified protein was identified by Western Blot.3) We prepared the rabbit polyclonal antibody of MCR protein and used it for Western Blot analysis, our results further confirms that the heterologously expressed protein was MCR. (4) By using hanging-drop vapor diffusion method with different kinds of protein crystallization kits to screen the initial crystallization condition of MCR, and after optimization the condition of crystal growth, we successfully obtained the 3-D single crystals. Some of them diffract X-ray to 3 A. In general,our results provide a robust basis for the further study of the structure and function of MCR.