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: Future studies of the mechanism of action of the mutants described in this paper would be significantly facilitated by access to an experimental 3D structure of XylS. In the absence of such data a homology‐based 3D model of the NTD will be useful, and we have therefore constructed such a model (Fig. 4). This was done by using DeepView (Guex and Peitsch, 1997) and the SwissModel server (Schwede et al., 2003), based on structure data from the RCSB Protein Data Bank (Berman et al., 2000). Evaluation of model quality was done with Errat (Colovos and Yeates, 1993), and the model predicts that the NTD consists of a β‐barrel connected via a loop region to two adjacent alpha helices forming a coiled coil. Based on comparison with the solved AraC structure (Soisson et al., 1997) and the observation that leucines 193 and 194 in XylS are involved in dimerization (Ruíz et al., 2003), we propose that the β‐barrel contains the inducer recognition pocket and that the coiled coil defines the dimerization surface. The substitutions identified in this work are therefore positioned in different structural sub‐motifs, and this leads to the hypothesis that they affect different functional aspects of the activator. It is difficult to conclude what specific functional role the different substituted amino acids might have, but according to the model it seems likely that R45 and A111 can interact with the inducer binding pocket. We have also carried out a separate alignment between XylS and other members of this family of transcriptional regulators (data not shown), and the results indicated that the amino acid substitutions reported here generally did not correlate with conserved residues within the family. Thus, the mutations appear to mainly represent XylS‐specific adaptations.
Affiliation: Department of Biotechnology, Norwegian University of Science and Technology, 7491 Trondheim, Norway.