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PEDS Advance Access originally published online on November 21, 2008
Protein Engineering Design and Selection 2009 22(2):53-58; doi:10.1093/protein/gzn069
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Evolving Lac repressor for enhanced inducibility

O. Satya Lakshmi and N.M. Rao1

Center for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500007, India

1 To whom correspondence should be addressed. E-mail: madhu{at}ccmb.res.in

Lactose repressor (LacI) is one of the best studied prokaryotic transcriptional regulatory proteins till date. Detailed structural, biochemical and genetic studies are being carried out on LacI since four decades to understand its ligand binding properties and the basis of allosteric response. We applied directed evolution methods on LacI to generate mutants with altered allosteric properties. After testing several hosts and expression vectors, a robust expression and screening system was optimised for identifying LacI variants with altered allosteric properties. After two rounds of error prone PCR (polymerase chain reaction) and shuffling, four mutants were selected from several thousand mutants, for their ability to induce reporter gene expression at 1 µM of isopropyl β-D-1-thiogalactopyranoside (IPTG). The observed combination of mutations in these four improved LacIs was not reported earlier. The mutant Lac repressors seem to operate as very good molecular switches by inducing gene expression at 1 µM of IPTG and confer 2–10 times higher level of gene expression as compared with the WT (wild -type).

Keywords: allostery/directed evolution/inducer/LacI/operator

Received June 9, 2008; revised October 18, 2008; accepted October 22, 2008.


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[Abstract] [Full Text] [PDF]


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