A single mutation in the core domain of the lac repressor reduces leakiness
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- 作者：Pietro Gatti-Lafranconi (1)
Willem P Dijkman (1)
Sean RA Devenish (1)
Florian Hollfelder (1) - 关键词：Lactose repressor ; Protein engineering ; Mutagenesis ; Differential scanning fluorimetry ; Protein production ; Synthetic biology ; Gene regulation ; LacI
- 刊名：Microbial Cell Factories
- 出版年：2013
- 出版时间：December 2013
- 年：2013
- 卷：12
- 期：1
- 全文大小：1,057 KB
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Willem P Dijkman (1)
Sean RA Devenish (1)
Florian Hollfelder (1)
1. Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK - ISSN：1475-2859
文摘
Background The lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression. In Nature, the ability to detect and respond to environmental conditions comes at the cost of the encoded enzymes being constitutively expressed at low levels. The readily-switched regulation provided by LacI has resulted in its widespread use for protein overexpression, and its applications in molecular biology represent early examples of synthetic biology. However, the leakiness of LacI that is essential for the natural function of the lac operon leads to an increased energetic burden, and potentially toxicity, in heterologous protein production. Results Analysis of the features that confer promiscuity to the inducer-binding site of LacI identified tryptophan 220 as a target for saturation mutagenesis. We found that phenylalanine (similarly to tryptophan) affords a functional repressor that is still responsive to IPTG. Characterisation of the W220F mutant, LacIWF, by measuring the time dependence of GFP production at different IPTG concentrations and at various incubation temperatures showed a 10-fold reduction in leakiness and no decrease in GFP production. Cells harbouring a cytotoxic protein under regulatory control of LacIWF showed no decrease in viability in the early phases of cell growth. Changes in responsiveness to IPTG observed in vivo are supported by the thermal shift assay behaviour of purified LacIWF with IPTG and operator DNA. Conclusions In LacI, long-range communications are responsible for the transmission of the signal from the inducer binding site to the DNA binding domain and our results are consistent with the involvement of position 220 in modulating these. The mutation of this single tryptophan residue to phenylalanine generated an enhanced repressor with a 10-fold decrease in leakiness. By minimising the energetic burden and cytotoxicity caused by leakiness, LacIWF constitutes a useful switch for protein overproduction and synthetic biology.