Recombinant Expression And Purification Of Magnesium Chelatase And Magnesium Protoporphyrin Methyltransferase From Rhodobacter Sphaeroides

Magnesium chelatase lies at the branch point of the heme and chlorophyll biosyntheticpathways，which is located at the branchpoint of tetrapyrrole biosynthesis. The first step isunique in chlorophyll formation, the insertion of Mg into protoporphyrin IX. This step iscatalyzed by magnesium chelatase and it is a main control point which directly affects thechlorophyll metabolism. The enzyme complex consists of three subunits, designated D, I,and H. Magnesium protoporphyrin methyltransferase is able to catalyze methylation ofMagnesium protoporphyrin IX to form magnesium protoporphyrin IX monomethylesterand it is the second enzyme in the chlorophyll biosynthetic pathways. Rhodobactersphaeroides not only plays an important role in the research of photosynthesis bacteria butalso has a certain reference value for researching on plant photosynthesis mechanism。In this study, first, we cloned four genes from Rhodobacter sphaeroide, whichparticipates in chlorophyll synthesis. Then, we analysed of the four genes expression andpurification in E. coli. The main results are summarized as follows:1. We cloned four genes BchM、BchI、BchD、BchH from Rhodobacter sphaeroide.Sequencing analysis confirmed the sequence has a small number of nucleotide differentfrom the login Rhodobacter sphaeroides sequences, compared with NCBI Blast.2. Four genes digested with two restrictive enzymes were inserted into two expressionvectors, respectively.3. Choose the optimal induced temperature when the protein expression. Four proteinsof molecular weight same as the expected, BchM is27kDa, BchI is40kDa, BchD is70kDa and BchH is140kDa.4. Purified recombinant protein BchM, electrophoresis analysis showed that the proteinwas pure.5. The C-terminal domain of the BchH was expressed and purified.In conclusion，we cloned genes encoding BchM, BChH, BChI and BChD, fromRhodobacter sphaeroides and analysis of their expression in E.coli. We purifiedrecombinant protein BchM、BchHC and laid the foundation for future research BchMstructure. These studies are in preparation for the study of chlorophyll biosyntheticpathway in E. coli.