A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression

The cell-free protein synthesis (CFPS) that synthesizes mRNA and protein from a template DNA has been featured as an important tool to emulate living systems in vitro. However, an obstacle to emulate living cells by CFPS is the loss of activity in the case of usage of high concentration cell extracts. In this study, we found that a high concentration of NTP which inhibits in the case of lower concentration cell extract restored the loss of CFPS activity using high concentration cell extracts. The NTP restoration was independent of the energy regeneration system used, and NTP derivatives also restored the levels of CFPS using a high concentration cell extract. Experiments using dialysis mode of CFPS showed that continuous exchange of small molecule reduced levels of NTP requirement and improved reaction speed of CFPS using the high concentration of cell extract. These findings contribute to the development of a method to understand the condition of living cells by in vitro emulation, and are expected to lead to the achievement of the reconstitution of living cells from biomolecule mixtures.

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

Methods and Protocols ◽

10.3390/mps2030068 ◽

2019 ◽

Vol 2 (3) ◽

pp. 68 ◽

Cited By ~ 5

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.

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Immobilization of Proteins of Cell Extract to Hydrogel Networks Enhances the Longevity of Cell-Free Protein Synthesis and Supports Gene Networks

ACS Synthetic Biology ◽

10.1021/acssynbio.0c00541 ◽

2021 ◽

Vol 10 (4) ◽

pp. 749-755

Author(s):

Xiaofei Ouyang ◽

Xiaoyu Zhou ◽

Sze Nga Lai ◽

Qi Liu ◽

Bo Zheng

Keyword(s):

Protein Synthesis ◽

Gene Networks ◽

Cell Extract ◽

Free Protein ◽

Cell Free Protein Synthesis

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

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00413-2 ◽

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.

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In vitro generation of anti-hepatitis B monoclonal antibodies from a single plasma cell using single-cell RT-PCR and cell-free protein synthesis

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2009.07.001 ◽

2010 ◽

Vol 109 (1) ◽

pp. 75-82 ◽

Cited By ~ 6

Author(s):

Yunita Sabrina ◽

Muhamad Ali ◽

Hideo Nakano

Keyword(s):

Protein Synthesis ◽

Monoclonal Antibodies ◽

Hepatitis B ◽

Single Cell ◽

Plasma Cell ◽

Rt Pcr ◽

Free Protein ◽

Cell Free Protein Synthesis ◽

Single Plasma

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Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.05.034 ◽

2004 ◽

Vol 319 (2) ◽

pp. 671-676 ◽

Cited By ~ 3

Author(s):

Nobutaka Nakashima ◽

Tomohiro Tamura

Keyword(s):

Protein Synthesis ◽

Pseudomonas Fluorescens ◽

Cell Extract ◽

Free Protein ◽

Cspa Promoter ◽

Cell Free Protein Synthesis

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The ovotestis of Aplysia californica: anatomy and egg release

Canadian Journal of Zoology ◽

10.1139/z80-304 ◽

1980 ◽

Vol 58 (12) ◽

pp. 2220-2229 ◽

Cited By ~ 7

Author(s):

F. Edward Dudek ◽

Hampik S. Injeyan ◽

Bonnie Soutar ◽

Greg Weir ◽

Stephen S. Tobe

Keyword(s):

Time Course ◽

Aplysia Californica ◽

Cell Extract ◽

Continuous Exposure ◽

Gradual Reduction ◽

Release Process ◽

Cell Extracts ◽

High K ◽

Scanning Electron

Egg release from the ovotestis of Aplysia californica has been studied using ovotestis fragments and bag cell extracts. Light and scanning electron microscopy showed clusters of follicles surrounded by muscle cells. Mature oocytes observed in egg masses and those released from ovotestis fragments were 90 μm in diameter. The number of mature releasable oocytes was relatively constant throughout the ovotestis, although a gradual reduction occurred with increasing distance from the small hermaphroditic duct.Bag cell induced egg release was detectable in vitro within 30 min and was complete by 180 min. The time course of egg release was similar under conditions of either continuous exposure or a 30-min pulse of bag cell extract. Artificial seawater (ASW) solutions with high K+ (110 mM) did not stimulate egg release unless bag cell extract was present. ASW with no Ca2+ and 3 mM EGTA or ASW containing Co2+ (10 mM) inhibited both bag cell induced and spontaneous (ASW alone) egg release.Therefore, brief exposure to bag cell peptide can trigger the egg release process, which is long lasting (~ 3 h) and Ca2+ dependent. The observation that high K+ did not stimulate egg release challenges the muscle contraction hypothesis of egg release.

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Functional and structural differences between photosynthetic and heterotrophic Rhodospirillum rubrum ribosomes and S-100 fractions

Canadian Journal of Microbiology ◽

10.1139/m76-225 ◽

1976 ◽

Vol 22 (10) ◽

pp. 1522-1539 ◽

Cited By ~ 1

Author(s):

C. T. Chow

Keyword(s):

Rhodospirillum Rubrum ◽

Photosynthetic Pigment ◽

Free Protein ◽

E Coli ◽

Highly Active ◽

Structural Differences ◽

S 100 ◽

Cell Free Protein Synthesis ◽

Fraction Addition

Cell-free, protein-synthesizing activity has been tested by using various combinations of the S-100 and ribosome fractions prepared from photosynthetic and heterotrophic Rhodospirillum rubrum. The photosynthetic ribosomes are highly active when combined with either the photosynthetic or the heterotrophic S-100 fractions, whereas the heterotrophic ribosomes are active only when combined with the photosynthetic S-100 fraction. Addition of a photosynthetic pigment-containing fraction to the homologous heterotrophic system is, however, able to stimulate its activity. An inhibitor and an activator involved in cell-free protein synthesis have been isolated from the stationary heterotrophic cells. The inhibitor is a very small, dialyzable compound which inhibits not only the R. rubrum but also the E. coli protein-synthesizing activity in vitro, whereas the activator is a non-dialyzable, small RNA molecule capable of stimulating only the R. rubrum activity. Differences exist between the photosynthetic and the heterotrophic systems in their response to various chemical compounds and to light as well as in their structure.

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