Escherichia coli Cell-Free Protein Synthesis and Isotope Labeling of Mammalian Proteins

2015 ◽  
pp. 311-345 ◽  
Author(s):  
Takaho Terada ◽  
Shigeyuki Yokoyama
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Isotope Labeling ◽  
Coli Cell ◽  
Free Protein ◽  
Mammalian Proteins ◽  
Cell Free Protein Synthesis

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

2019 ◽  
Vol 2 (3) ◽  
pp. 68 ◽  
Author(s):  
Kim ◽  
Copeland ◽  
Padumane ◽  
Kwon
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Optimization Procedure ◽  
Cell Extract ◽  
Coli Cell ◽  
Free Protein ◽  
E Coli ◽  
Highly Active ◽  
Cell Free Protein Synthesis

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.

scholarly journals Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins

2019 ◽  
Vol 2 (2) ◽  
pp. 28 ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Cell Killing ◽  
Great Promise ◽  
Immunity Protein ◽  
Free Protein ◽  
Killing Activity ◽  
Colicin E1 ◽  
Colicin E3 ◽  
Cell Free Protein Synthesis

Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are effective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we confirmed our previous finding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our findings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform.

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