title: Directed evolution of efficient secretion in the SRP-dependent export of TolB.
abstract: Signal sequence non-optimal codons have been shown to be important for the folding and efficient export of maltose binding protein (MBP), a SecB dependent protein. In this study, we analysed the importance of signal sequence non-optimal codons of TolB, a signal recognition particle (SRP) dependent exported protein. The protein production levels of wild type TolB (TolB-wt) and a mutant allele of TolB in which all signal sequence non-optimal codons were changed to a synonymous optimal codon (TolB-opt), revealed that TolB-opt production was 12-fold lower than TolB-wt. This difference could not be explained by changes in mRNA levels, or plasmid copy number, which was the same in both strains. A directed evolution genetic screen was used to select for mutants in the TolB-opt signal sequence that resulted in higher levels of TolB production. Analysis of the 46 independent TolB mutants that reverted to wild type levels of expression revealed that at least four signal sequence non-optimal codons were required. These results suggest that non-optimal codons may be required for the folding and efficient export of all proteins exported via the Sec system, regardless of whether they are dependent on SecB or SRP for delivery to the inner membrane.
authors.affiliation: Department of Microbiology and Immunology, Emory University, Atlanta, GA 30322, USA.
authors.forename: Yaramah M
authors.initials: YM
authors.lastname: Zalucki
authors.forename: William M
authors.initials: WM
authors.lastname: Shafer
authors.forename: Michael P
authors.initials: MP
authors.lastname: Jennings
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D003062:Codon; D017931:DNA Primers; D019020:Directed Molecular Evolution; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018271:Signal Recognition Particle / Q000502:physiology*
chemical_list: D003062:Codon; D017931:DNA Primers; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; D018271:Signal Recognition Particle; C062094:tolB protein, E coli