Improving recombinant membrane protein overexpression in Escherichia coli

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, I have designed a selection strategy to isolate mutant strains of E. coli that improve the expression of a targeted membrane protein. Using this novel method, I have isolated mutant strains with up to 75-fold increased expression. The mutant strains also prove to be an advance over the current standards for membrane protein production, the strains C41(DE3) and C43(DE3).;I have characterized two of the improved membrane protein expression strains using an approach that is a hybrid of classical genetics and whole genome sequencing. Classical methods have indicated that both strains contain at least two phenotype-conferring mutations and have highlighted the possible locations of these mutations. Whole genome sequencing has identified all genomic differences between the wild-type strain and the evolved strains.;I have also characterized a third mutant found to have the phenotype of low plasmid copy number in addition to its improved expression phenotype. This additional trait has fortuitously allowed me to rationally identify the source of the improvement. A truncation of the gene pcnB encoding poly (A) polymerase is likely to improve membrane protein expression by decreasing plasmid copy number or increasing mRNA stability, or both.