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Heterologous Expression of Membrane‐Protein Subunits in E. coli: The Subunit B of the Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)

The PmoB subunit of particulate methane monooxygenase (pMMO) from M. capsulatus (Bath) exhibits strong affinity towards reduced copper ions. To manifest this consequence, we translocated this subunit into the membranes of E. coli TB1 by molecular biology techniques. The success of recombinant was evidenced by western blotting with the detection of the MBP fusion‐protein of the PmoB. Gel electrophoresis also gave the correct molecular‐weight pattern in the 1D SDS PAGE. Subsequently, we transformed the designed vector with the pmoB insert into a strain BCRC®50305 that belongs to one of the E. coli strains originated from the copper tolerant species W3110. This strain of bacteria could be grown in a medium containing up to 3.1 mM CuSO4(aq). We subjected this bacterial strain with the pmoB gene to grow in LB buffer containing 1.0 mM Cu ions. Under high copper stress, the cellular membranes of E. coli behave like the ones in M. capsulatus with abundant membrane accumulation and high reduced copper content. These results suggest that both M. capsulatus and BCRC®50305 bear similar facilities for membrane production and cellular copper ions incorporation. With better control over copper homeostasis, BCRC®50305 would allow the subunit B of pMMO to attain maturation of the membrane‐bound subunit with the correct folding.