Directed evolution of the transcription factor XylS for development of improved expression systems.
Bottom Line: Here we report directed evolution of XylS resulting in mutant proteins with increased ability to stimulate transcription in Escherichia coli from Pm.Through in silico 3D modelling of the N-terminal domain of XylS, it was observed that the evolved mutant proteins contained substitutions that were positioned in different parts of the predicted structure, including a β-barrel putatively responsible for effector binding and a coiled coil probably important for dimerization.The total production of the host-toxic antibody fragment scFv-phOx expressed from Pm with the evolved XylS mutant protein StEP-13 was about ninefold higher than with wild-type XylS, demonstrating that directed evolution of transcription factors can be an important new tool to achieve high-level recombinant protein production.
Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway.Show MeSH
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Mentions: The mutant library was screened for increased ampicillin tolerance under inducing conditions, and 40 different xylS variants were identified. Among them 14 different xylS point mutations assumed to be at least partly responsible for the observed phenotypes were identified. These mutations were identified because of their repeated occurrence or because they were the only mutations present. The corresponding amino acid sequence changes were all positioned in the NTD of the XylS protein. To verify the assumption that these substitutions increase the ability of XylS to stimulate transcription from Pm, site‐specific mutagenesis was used to construct 14 different xylS variants containing each of the identified point mutations. All the new xylS variants increased their host cells' tolerance to ampicillin compared with cells with wild‐type xylS (Fig. 2).
Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway.