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Updated: Jun 20, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Engineering and directed evolution of a Ca2+ binding site A-deficient AprE mutant reveal an essential contribution of
Eliel R Romero-García1, Alfredo Téllez-Valencia, María F Trujillo
1Departamento de Biología, Universidad de Guanajuato, Colonia Noria Alta S/N, Guanajuato, 36050 Guanajuato, Mexico.
Abstract:
An aprE mutant from B. subtilis 168 lacking the connecting loop Leu(75)-Leu(82) which is predicted to encode a Ca(2+) binding site was constructed. Expression of the mutant gene (aprEDeltaLeu(75)-Leu(82)) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEDeltaL(75)-L(82). An AprEDeltaL(75)-L(82) variant with reestablished enzyme activity was selected by directed evolution. The novel mutations Thr(66)Met/Gly(102)Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEDeltaL(75)-L(82) T(66)M G(102)D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects beta-sheet e3 with alpha-helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.
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