PMID: 11342710May 9, 2001Paper

Different strategies to recover the activity of monomeric triosephosphate isomerase by directed evolution

Protein Engineering
G Saab-RincónX Soberón

Abstract

A monomeric version of triosephosphate isomerase from Trypanosoma brucei, MonoTIM, has very low activity, and the same is true for all of the additional monomeric variants so far constructed. Here, we subjected MonoTIM to directed evolution schemes to achieve an activity improvement. The construction of a suitable strain for genetic selection provided an effective way to obtain active catalysts from a diverse population of protein variants. We used this tool to identify active mutants from two different strategies of mutagenesis: random mutagenesis of the whole gene and randomization of loop 2. Both strategies converged in the isolation of mutations Ala43 to Pro and Thr44 to either Ala or Ser, when randomizing the entire gene or to Arg in the case of randomization of loop 2. The kinetic characterization of the two more active mutants showed an increase of 11-fold in k(cat) and a reduction of 4-fold in K(m) for both of them, demonstrating the sensitivity of the selection method. A small difference in growth rate is observed when both mutant genes are compared, which seems to be attributable to a difference in solubility of the expressed proteins.

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Citations

Apr 29, 2005·Biomolecular Engineering·Birte Höcker
Sep 25, 2002·Journal of Molecular Biology·Adela Rodríguez-RomeroD Alejandro Fernández-Velasco
Aug 10, 2010·Cellular and Molecular Life Sciences : CMLS·R K WierengaR Venkatesan
Aug 23, 2006·Critical Reviews in Biotechnology·Jasjeet Kaur, Rohit Sharma
Feb 13, 2016·PeerJ·Edson N Carcamo-Noriega, Gloria Saab-Rincon
Jul 24, 2007·Biochimica Et Biophysica Acta·Hugo NájeraD Alejandro Fernández-Velasco
Mar 24, 2009·Biochemical and Biophysical Research Communications·Francisco Zárate-PérezEdgar Vázquez-Contreras
Mar 15, 2015·Protein Engineering, Design & Selection : PEDS·Mirja KrauseRik K Wierenga
Apr 30, 2019·The Plant Journal : for Cell and Molecular Biology·Eduardo Castro-TorresLuis G Brieba
Nov 9, 2007·PLoS Neglected Tropical Diseases·Vanesa Olivares-IllanaArmando Gómez-Puyou
May 2, 2002·The Journal of Biological Chemistry·Xiangduo KongJin Zhang
Nov 10, 2005·Chemical Reviews·Reinhard Sterner, Birte Höcker

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