scholarly journals Translational incorporation of modified phenylalanines and tyrosines during cell-free protein synthesis

RSC Advances ◽  
2020 ◽  
Vol 10 (19) ◽  
pp. 11013-11023
Author(s):  
Zhongqiang Wang ◽  
Hayden Matthews
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Wild Type ◽  
Free Protein ◽  
Translational Machinery ◽  
E Coli ◽  
Cell Free Protein Synthesis

E. coli wild-type translational machinery utilizes a range of nonproteinogenic amino acids for protein synthesis with incorporation levels greater than 95%.

scholarly journals Development of an E. coli strain for cell-free ADC manufacturing

2021 ◽  
Author(s):  
Dan Groff ◽  
Nina Carlos ◽  
Rishard Chen ◽  
Jeff Hanson ◽  
Shengwen Liang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Large Scale ◽  
Continuous Fermentation ◽  
Free Protein ◽  
E Coli ◽  
Natural Amino Acids ◽  
Synthesis Reactions ◽  
Cell Free Protein Synthesis

Recent advances in cell-free protein synthesis have enabled the folding and assembly of full-length antibodies at high titers with extracts from prokaryotic cells. Coupled with the facile engineering of the E. coli translation machinery, E. coli based in vitro protein synthesis reactions have emerged as a leading source of IgG molecules with non-natural amino acids incorporated at specific locations for producing homogeneous antibody drug conjugates. While this has been demonstrated with extract produced in batch fermentation mode, continuous extract fermentation would facilitate supplying material for large-scale manufacturing of protein therapeutics. To accomplish this, the IgG-folding chaperones DsbC and FkpA, and orthogonal tRNA for non-natural amino acid production were integrated onto the chromosome with high strength constitutive promoters. This enabled co-expression of all three factors at a consistently high level in the extract strain for the duration of a five-day continuous fermentation. Cell-free protein synthesis reactions with extract produced from cells grown continuously yielded titers of IgG containing non-natural amino acids above those from extract produced in batch fermentations. In addition, the quality of the synthesized IgGs and the potency of ADC produced with continuously fermented extract were indistinguishable from those produced with batch extract. These experiments demonstrate that continuous fermentation of E. coli to produce extract for cell-free protein synthesis is feasible and helps unlock the potential for cell-free protein synthesis as a platform for biopharmaceutical production.

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

Interfacing a Personal Glucose Meter with Cell-Free Protein Synthesis for Rapid Analysis of Amino Acids

2019 ◽  
Vol 91 (3) ◽  
pp. 2531-2535 ◽  
Author(s):  
Yeon-Jae Jang ◽  
Kyung-Ho Lee ◽  
Tae Hyeon Yoo ◽  
Dong-Myung Kim
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Rapid Analysis ◽  
Free Protein ◽  
Personal Glucose Meter ◽  
Glucose Meter ◽  
Cell Free Protein Synthesis

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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