Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

Jonghyeon Shin,Vincent Noireaux

doi:10.1186/1754-1611-4-8

Abstract

BackgroundEscherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Recently, informational processes have been reconstituted in vitro with cell-free systems. These synthetic approaches, however, have been seriously limited by a lack of transcription modularity. The current available cell-free systems have been optimized to work with bacteriophage RNA polymerases, which put significant restrictions to engineer processes related to biological information. The development of efficient cell-free systems with broader transcription capabilities is required to study complex informational processes in vitro.ResultsIn this work, an efficient cell-free expression system that uses the endogenous E. coli RNA polymerase only and sigma factor 70 for transcription was prepared. Approximately 0.75 mg/ml of Firefly luciferase and enhanced green fluorescent protein were produced in batch mode. A plasmid was optimized with different regulatory parts to increase the expression. In addition, a new eGFP was engineered that is more translatable in cell-free systems than the original eGFP. The protein production was characterized with three different adenosine triphosphate (ATP) regeneration systems: creatine phosphate (CP), phosphoenolpyruvate (PEP), and 3-phosphoglyceric acid (3-PGA). The maximum protein production was obtained with 3-PGA. Preparation of the crude extract was streamlined to a simple routine procedure that takes 12 hours including cell culture.ConclusionsAlthough it uses the endogenous E. coli transcription machinery, this cell-free system can produce active proteins in quantities comparable to bacteriophage systems. The E. coli transcription provides much more possibilities to engineer informational processes in vitro. Many E. coli promoters/operators specific to sigma factor 70 are available that form a broad library of regulatory parts. In this work, cell-free expression is developed as a toolbox to design and to study synthetic gene circuits in vitro.

Highlights

Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins
E. coli cell-free extracts working with the endogenous core RNA polymerase and the housekeeping sigma factor 70 were originally prepared to study gene expression mechanisms [19,20]
The protein production with this extract is comparable to bacteriophage systems

Summary

Introduction

Escherichia coli cell-free expression systems use bacteriophage RNA polymerases, such as T7, to synthesize large amounts of recombinant proteins. These systems are used for many applications in biotechnology, such as proteomics. Informational processes have been reconstituted in vitro with cell-free systems These synthetic approaches, have been seriously limited by a lack of transcription modularity. Reconstitution of cell-free informational processes, and on a broader range the field of in vitro synthetic biology, is limited to bacteriophage RNA polymerases [12,13,14,15,16,17]. The reconstitution of informational processes in vitro would greatly benefit from having efficient cell-free systems that offer a greater modularity at the level of transcription. Bacteriophage transcription has been largely favored at the expense of endogenous transcription systems

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Conclusion