A stabilized TEM-1 β-lactamase as a scaffold for engineering allosteric enzymes
(2013)
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- Authors
- Supervisors
- Soumillion, Patrice
- Abstract
- (en) The design of proteins with desired properties through genetic engineering is generally restricted by the mutations tolerance of the starting scaffold. Gain in thermostability is known to increase the robustness of proteins toward substitutions. With the aim of generating allosteric enzymes selectable by phage display, we managed to highly improve the tolerance of a TEM-1 β-lactamase toward loop insertional mutagenesis upon stabilization. A highly diverse collection of mutants endowed with complex potential binding sites was built by insertion of random peptides and mutations in three contiguous surface loops near the active site. Stabilization increased the number of active triple mutants (6.6 x 107) by 70 fold. The gain in stability also abolished the requirement for a stabilizing disulfide bridge in the degenerated N-terminal loop. This increase in diversity is believed to promote the selection of clones with new binding abilities that might predispose the protein to exhibit allosteric effects. Twelve clones recognizing nickel were selected by phage display and 25% of them showed allosteric regulation upon metal ions binding. The enzyme prestabilization along with a possible stiffening of the scaffold did not seem to alter the selection of allosteric clones. Besides, the insertions of the stabilizing mutations in a previously described allosteric clone activated by aminoglycosides had no impact on the regulation properties of the enzyme. These results validate the choice of the stabilized TEM-1 β-lactamase as a scaffold for engineering allosteric enzymes.
- Affiliations
Citations
Baudoux, B. (2013). A stabilized TEM-1 β-lactamase as a scaffold for engineering allosteric enzymes. https://hdl.handle.net/2078.5/203608