scholarly journals Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Yong-Chan Kwon ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Host Cells ◽  
Persister Cells ◽  
Free Protein ◽  
E Coli ◽  
Dna And Rna ◽  
Rapid Production ◽  
Cell Free Protein Synthesis

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

scholarly journals Biosynthesis of Escherichia coli adenosine deaminase using cell-free protein synthesis

2021 ◽  
Vol 66 (3) ◽  
pp. 271-276
Author(s):  
I. S. Kazlouski ◽  
A. I. Zinchenko
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Adenosine Deaminase ◽  
Submerged Cultivation ◽  
Process Conditions ◽  
Experimental Sample ◽  
Free Protein ◽  
E Coli ◽  
Cell Free Protein Synthesis

One of the recent perspective trends of molecular biotechnology is cell-free protein synthesis (CFPS). The procedure of CFPS is based on in vitro reconstruction of all stages of a biosynthesis of protein in a whole cell, including a transcription, an aminoacylation of tRNA and translation of mRNA by ribosomes.Previously, we constructed a strain Escherichia coli that produces homologous adenosine deaminase (ADase). In the present study, as an alternative to canonical submerged cultivation in a fermenter, the possibility of the ADase synthesis in the system of CFPS was studied. For synthesis of this enzyme we used the E. coli-30 cell extract, T7 bacteriophage RNA polymerase, and high-copy plasmid vector pET42mut with gene ADase inserted into it.As a result of the work we have demonstrated for the first time the possibility of synthesis of ADase E. coli in the CFPS system. In a partially optimized process conditions, an experimental sample of recombinant AD with an activity of 530 U/ml of enzyme preparation was obtained.

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 Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Liyuan Zhang ◽  
Xiaomei Lin ◽  
Ting Wang ◽  
Wei Guo ◽  
Yuan Lu
Keyword(s):  
Protein Synthesis ◽  
Protein Production ◽  
Influential Factors ◽  
Competent Cell ◽  
Free Protein ◽  
Application Range ◽  
E Coli ◽  
Short Time ◽  
Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

Author response for "A rational approach to improving titer in E. coli ‐based cell‐free protein synthesis reactions"

2020 ◽  
Author(s):  
Noelle Colant ◽  
Beatrice Melinek ◽  
Jaime Teneb ◽  
Stephen Goldrick ◽  
William Rosenberg ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Author Response ◽  
Rational Approach ◽  
Free Protein ◽  
E Coli ◽  
Synthesis Reactions ◽  
Cell Free Protein Synthesis

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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