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
Related in: MedlinePlus
Mentions: To test whether different substitutions in XylS can be combined to yield activators that further increase transcription from Pm, new xylS genes encoding activators containing two or three substitutions were constructed by site‐specific mutagenesis. This included combinations of substitutions identified after error‐prone PCR and the R45T substitution, which has previously been shown to increase XylS activity (Ramos et al., 1990). Eighteen different combinations were tested, and all of them gave their hosts higher induced ampicillin tolerance than mutants with single mutations in xylS (Fig. 3), verifying that the XylS substitutions can act in an additive manner. The XylS protein Syn‐15 (containing the substitutions F3Y, I50T and F97L) gave about sixfold higher induced ampicillin tolerance than wild type, and only 1.5‐fold higher basal ampicillin tolerance, thus increasing the induction ratio from 17 for wild‐type XylS to at least 56 with Syn‐15.
Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway.