Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery

Marina Snapyan,Vehary Sakanyan,Sylvain Robin,Garabet Yeretssian,Frédéric Marc,Michèle Lecocq

doi:10.3390/biotech10040024

Abstract

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

Highlights

A cell-free method for protein production has been successfully implemented by translating exogenously added nucleic acid into cell extracts of Escherichia coli [1]
In order to gain higher gene expression from the pargC promoter, we decided to increase the pool of the bacterial RNA polymerase in the reaction mixture by using the cell-free extracts in which core enzyme subunits were overexpressed
Higher gene expression from the pargC promoter, we decided to increase the pool of the bacterial RNA polymerase in the reaction mixture by using the cell‐free extracts in which core enzyme subunits were overexpressed

Summary

Introduction

A cell-free method for protein production has been successfully implemented by translating exogenously added nucleic acid into cell extracts of Escherichia coli [1]. Protein synthesis using the T7 phage transcriptional machinery increased protein yield and turned cell-free protein synthesis (CFPS) into a powerful approach for studying the proteomes of organisms with sequenced genomes without performing laborious gene cloning in living cells. Similar CFPS systems have been created using other bacterial species to achieve additional advantages over the E. coli system for detecting specific protein–protein interactions and identifying potential inhibitors of desired proteins [4,5]. The protein yield depends on the rate of transcription and on the stability of the target mRNAs

Methods

Results

Discussion

Conclusion