A positive feedback-based gene circuit to increase the production of a membrane protein

Karan Bansal,Ke Yang,Kaustubh D Bhalerao,Goutam J Nistala,Robert B Gennis

doi:10.1186/1754-1611-4-6

Abstract

BackgroundMembrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. However, membrane proteins are often difficult to produce in large quantities for the purpose of crystallographic or biochemical analyses.ResultsIn this paper, we demonstrate that synthetic gene circuits designed specifically to overexpress certain genes can be applied to manipulate the expression kinetics of a model membrane protein, cytochrome bd quinol oxidase in E. coli, resulting in increased expression rates. The synthetic circuit involved is an engineered, autoinducer-independent variant of the lux operon activator LuxR from V. fischeri in an autoregulatory, positive feedback configuration.ConclusionsOur proof-of-concept experiments indicate a statistically significant increase in the rate of production of the bd oxidase membrane protein. Synthetic gene networks provide a feasible solution for the problem of membrane protein production.

Highlights

Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development
Precise regulation of gene expression either through exogenous control or through endogenous autoregulation is often critical for the development of synthetic biology applications
There are other luxR* pLuxI-luxR*-bd similar positive feedback circuits based on the wild type LuxR, which rely on the action of Acyl homoserine lactone (AHL), either exogenously supplied, or produced by the action of luxI, for their expression system to operate correctly

Summary

Introduction

Membrane proteins are an important class of proteins, playing a key role in many biological processes, and are a promising target in pharmaceutical development. Precise regulation of gene expression either through exogenous control or through endogenous autoregulation is often critical for the development of synthetic biology applications. A positive feedback within the regulatory circuit can lead to increased steady state level (amplification) of the gene expression, faster response kinetics, and increased sensitivity to exogenous inducers or autoinducers [16,17,18,19]. These properties are suitable for applications such as protein production, where the benefits of increased production rates can directly translate into reduced resource costs and increased profits

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Results

Conclusion