scholarly journals Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Keita Hibi ◽  
Kazuaki Amikura ◽  
Naoki Sugiura ◽  
Keiko Masuda ◽  
Satoshi Ohno ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Genetic Code ◽  
Active Protein ◽  
Free System ◽  
Free Protein ◽  
Pure System ◽  
A Cell ◽  
Cell Free Protein Synthesis ◽  
Decoding Fidelity

AbstractEntire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

scholarly journals Identification of a gene that makes a protein by using a cell free protein synthesis system

2021 ◽  
Vol 8 (4) ◽  
pp. 124-125
Author(s):  
Umair Masood
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Living Cell ◽  
Molecular Size ◽  
Synthesis System ◽  
Free Protein ◽  
A Cell ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

A living cell could be genetically modified to perform a function such as the production of a protein. However, these genetic modifications often conflict with normal cellular function and result in a mutation. Defects can be overcome through removing the bacterial membrane which leaves the lysate that is performing both transcription and translation. The cell free-protein synthesis is also known as in vitro protein synthesis and is the production of a protein without using a living cell. The gene is acting as instructions to make the protein. If we can isolate a gene and then apply a cell free protein synthesis system after synthesis the protein and run on gel-electrophoresis we can identify a gene on the basis of the protein. Gel electrophoresis is a laboratory technique used to ______ contrasting proteins according to molecular size and charge.

scholarly journals A factor converting monoribosomes into polyribosomes during protein synthesis in vitro

1968 ◽  
Vol 110 (2) ◽  
pp. 231-236 ◽  
Author(s):  
Brian B. Cohen
Keyword(s):  
Protein Synthesis ◽  
Potassium Chloride ◽  
Active Component ◽  
Synthesis System ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described previously (Miller, Hamada, Yang, Cohen & Schweet, 1967). The participation of the extract in cell-free protein synthesis was studied. Purified polyribosomes were isolated and converted into monoribosomes by incubation in the cell-free protein-synthesis system. The monoribosomes were isolated and found to be unable to synthesize protein in the cell-free system. The addition of the ribosomal extract to the system stimulated protein synthesis. This was accompanied by the conversion of some of the monoribosomes into polyribosomes. The active component or components of the extract were shown to be protein.

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.

scholarly journals Directed Evolution of Proteins throughIn VitroProtein Synthesis in Liposomes

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Takehiro Nishikawa ◽  
Takeshi Sunami ◽  
Tomoaki Matsuura ◽  
Tetsuya Yomo
Keyword(s):  
Protein Synthesis ◽  
Directed Evolution ◽  
Single Copy ◽  
Functional Evaluation ◽  
Future Directions ◽  
Pure System ◽  
Protein Functions ◽  
A Cell ◽  
Evolution Of Proteins

Directed evolution of proteins is a technique used to modify protein functions through “Darwinian selection.”In vitrocompartmentalization (IVC) is anin vitrogene screening system for directed evolution of proteins. IVC establishes the link between genetic information (genotype) and the protein translated from the information (phenotype), which is essential for all directed evolution methods, by encapsulating both in a nonliving microcompartment. Herein, we introduce a new liposome-based IVC system consisting of a liposome, the protein synthesis using recombinant elements (PURE) system and a fluorescence-activated cell sorter (FACS) used as a microcompartment,in vitroprotein synthesis system, and high-throughput screen, respectively. Liposome-based IVC is characterized byin vitroprotein synthesis from a single copy of a gene in a cell-sized unilamellar liposome and quantitative functional evaluation of the synthesized proteins. Examples of liposome-based IVC for screening proteins such as GFP andβ-glucuronidase are described. We discuss the future directions for this method and its applications.

Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

2014 ◽  
Vol 19 (3) ◽  
pp. 426-432 ◽  
Author(s):  
Su-Jin Oh ◽  
Kyung-Ho Lee ◽  
Ho-Cheol Kim ◽  
Christy Catherine ◽  
Hyungdon Yun ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Unnatural Amino Acids ◽  
Synthesis System ◽  
Free Protein ◽  
A Cell ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis
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