Co-expression Of Dimethyl Sulfide Monooxygenase By Single Vector Dual Promoter Strategy

DMS monooxygenase consists of two subunits: DmoA and DmoB. DmoA is a 53 kDa FMNH2-dependent monooxygenase and DmoB is a 19 kDa NAD(P)H-dependent oxidoreductase. From previous research the gene sequence of dmoA is known; however, there are two sequence candidates for NAD(P)H-dependent oxidoreductases, dmoB136 and dmoB176, encoded on the dmo operon. Also, previous research suggests a strong interaction between subunits, providing precedence for co-expression studies. The dmoA gene and dmoB136 were cloned into a p-DUET plasmid with streptomycin resistance and a CDF replicon. The dmoB136 gene is located in multiple cloning site 1 containing a N-terminal a His6-tag, and the dmoA gene is located in multiple cloning site 2 followed by a C-terminal S-tag. The plasmid containing genes for both subunits was transformed in C41(DE3) E. coli and expressed. Purification strategies include affinity and size-exclusion chromatography. Initial data suggests co-expression of DmoA and DmoB as analyzed by SDS-PAGE. The presence of DmoB is further evidenced by initial activity assays, with higher activity in assays containing FMN as compared to those without FMN. Current research is being performed to optimize the purification strategies for isolating each subunit in order to test each for enzyme activity, kinetics, and cofactor requirements.