A Crude Extract Preparation and Optimization from a Genomically Engineered Escherichia coli for the Cell-Free Protein Synthesis System: Practical Laboratory Guideline.

Kim Kim,Copeland Copeland,Padumane Padumane,Kwon Kwon

doi:10.3390/mps2030068

Abstract

With the advancement of synthetic biology, the cell-free protein synthesis (CFPS) system has been receiving the spotlight as a versatile toolkit for engineering natural and unnatural biological systems. The CFPS system reassembles the materials necessary for transcription and translation and recreates the in vitro protein synthesis environment by escaping a physical living boundary. The cell extract plays an essential role in this in vitro format. Here, we propose a practical protocol and method for Escherichia coli-derived cell extract preparation and optimization, which can be easily applied to both commercially available and genomically engineered E. coli strains. The protocol includes: (1) The preparation step for cell growth and harvest, (2) the thorough step-by-step procedures for E. coli cell extract preparation including the cell wash and lysis, centrifugation, runoff reaction, and dialysis, (3) the preparation for the CFPS reaction components and, (4) the quantification of cell extract and cell-free synthesized protein. We anticipate that the protocol in this research will provide a simple preparation and optimization procedure of a highly active E. coli cell extract.

Highlights

The technology involving the disruption of bacterial cells and collection of ribosomes for synthesizing proteins was first introduced when the fraction of ribosomes was identified as the core of the protein synthesis machinery of the cells [1]
The cell-free protein synthesis (CFPS) system has been developed for exclusive protein synthesis utilizing active ribosomes and other cellular machinery outside of the living cell [2,3]
The CFPS system offers many advantages over a cell-based system, such as ease of manipulating biochemical pathways [6,7], higher tolerance on chemicals and toxic compounds [8,9], utilization of PCR amplified linear template allowing for high-throughput preparation and protein synthesis of the gene of interest and breadboarding synthetic biological circuit [10,11,12,13], and the capability of highly efficient non-standard amino acid incorporation [14,15]

Summary

Introduction

The technology involving the disruption of bacterial cells and collection of ribosomes for synthesizing proteins was first introduced when the fraction of ribosomes was identified as the core of the protein synthesis machinery of the cells [1]. The preparation step of the cell extract is crucial, as it contains key components for synthesizing proteins, so it is important to practice the optimized cell-free extract method for each E. coli strain for high protein yield. The recent study from Kwon and Jewett was the first to introduce an optimized systemic cell extract preparation process for the non-commercial engineered E. coli strain K12 MG1655 (C495) which greatly advanced the potential for use in future biomedical/industrial applications [22]. The systemically optimized CFPS is inspiring novel ways to utilize the cell extracts from engineered E. coli strains for applications involving unnatural amino acid incorporation [15], patient-specific therapeutic vaccines [24], anticancer protein production [25], and more

Methods

Results

Conclusion