scholarly journals Two fractions required for cell-free protein synthesis with components from rabbit reticulocytes

1969 ◽  
Vol 115 (3) ◽  
pp. 523-527 ◽  
Author(s):  
Brian B. Cohen
Keyword(s):  
Protein Synthesis ◽  
Active Component ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Analytical Ultracentrifuge ◽  
Active Components ◽  
Free Protein ◽  
Cell Free Protein Synthesis ◽  
Rabbit Reticulocytes

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described by Miller, Hamada, Yang, Cohen & Schweet (1967). This extract has been shown to convert monoribosomes into polyribosomes during protein synthesis in vitro (Cohen, 1968). The nature of this extract was studied in greater detail. Centrifugation of the extract through a sucrose density gradient separated the activity into a fast-sedimenting fraction. The two fractions were shown to have different functions in stimulating cell-free protein synthesis and their active components were shown to be protein or partly protein in nature. Each fraction was analysed by electrophoresis and in the analytical ultracentrifuge. It was concluded that the active component in the fast-sedimenting fraction had a sedimentation coefficient of 15·5s and that of the slow-sedimenting fraction 10·5s.

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 Hemin reversal of benzene-induced inhibition of reticulocyte protein synthesis

Blood ◽  
1976 ◽  
Vol 47 (1) ◽  
pp. 145-154 ◽  
Author(s):  
FJ Forte ◽  
HS Cohen ◽  
J Rosman ◽  
ML Freedman
Keyword(s):  
Protein Synthesis ◽  
Sickle Cell ◽  
Clinical Implications ◽  
Intact Cells ◽  
Free Protein ◽  
Translational Repressor ◽  
Heme Synthesis ◽  
Dependent Site ◽  
Cell Free Protein Synthesis ◽  
Rabbit Reticulocytes

Abstract Benzene (0.056–0.113 M) rapidly and reversibly inhibited protein synthesis in anucleate human sickle cell and rabbit reticulocytes. Hemin (50 muM) both prevented and reversed this effect of benzene. The inhibition in rabbit reticulocytes was accompanied by a conversion of polyribosomal disaggregation required ribosomal movement along mRNA and was also prevented and reversed by 50 muM hemin. Benzene was also shown to inhibit heme synthesis in rabbit reticulocytes while neither ATP nor GSH levels were altered. A translational repressor (HCR) of reticulocyte cell-free protein synthesis was isolated from intact cells incubated with benzene, while no significant amount of HCR was found in cells incubated with both benzene and hemin. These results indicated that benzene inhibits translation at the heme-dependent site of initiation. The clinical implications of these experiments remain to be elucidated.

scholarly journals In Vitro Reconstruction of Nonribosomal Peptide Biosynthesis Directly from DNA Using Cell-Free Protein Synthesis

2016 ◽  
Vol 6 (1) ◽  
pp. 39-44 ◽  
Author(s):  
Anthony W. Goering ◽  
Jian Li ◽  
Ryan A. McClure ◽  
Regan J. Thomson ◽  
Michael C. Jewett ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Nonribosomal Peptide ◽  
Free Protein ◽  
Peptide Biosynthesis ◽  
Cell Free Protein Synthesis

Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes

2008 ◽  
Vol 133 (2) ◽  
pp. 183-189 ◽  
Author(s):  
Mami Hino ◽  
Masatoshi Kataoka ◽  
Kazuaki Kajimoto ◽  
Takenori Yamamoto ◽  
Jun-Ichi Kido ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Wheat Germ ◽  
Cell Extract ◽  
Crude Cell Extract ◽  
Free Protein ◽  
Cell Free Protein Synthesis ◽  
Rabbit Reticulocytes

scholarly journals <italic>In vitro</italic> synthetic biology: Cell-free protein synthesis

2017 ◽  
Vol 62 (33) ◽  
pp. 3851-3860
Author(s):  
Yang LIU ◽  
XiaoCui GUO ◽  
JinHui GENG ◽  
Yi JIAO ◽  
JinPeng HAN ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Synthetic Biology ◽  
Free Protein ◽  
Cell Free Protein Synthesis

scholarly journals Cell-free prototyping of limonene biosynthesis using cell-free protein synthesis

2020 ◽  
Author(s):  
Quentin M. Dudley ◽  
Ashty S. Karim ◽  
Connor J. Nash ◽  
Michael C. Jewett
Keyword(s):  
Protein Synthesis ◽  
Metabolic Engineering ◽  
Biosynthetic Enzymes ◽  
List Type ◽  
Free Protein ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
The Impact ◽  
Cell Free Protein Synthesis

AbstractMetabolic engineering of microorganisms to produce sustainable chemicals has emerged as an important part of the global bioeconomy. Unfortunately, efforts to design and engineer microbial cell factories are challenging because design-built-test cycles, iterations of re-engineering organisms to test and optimize new sets of enzymes, are slow. To alleviate this challenge, we demonstrate a cell-free approach termed in vitro Prototyping and Rapid Optimization of Biosynthetic Enzymes (or iPROBE). In iPROBE, a large number of pathway combinations can be rapidly built and optimized. The key idea is to use cell-free protein synthesis (CFPS) to manufacture pathway enzymes in separate reactions that are then mixed to modularly assemble multiple, distinct biosynthetic pathways. As a model, we apply our approach to the 9-step heterologous enzyme pathway to limonene in extracts from Escherichia coli. In iterative cycles of design, we studied the impact of 54 enzyme homologs, multiple enzyme levels, and cofactor concentrations on pathway performance. In total, we screened over 150 unique sets of enzymes in 580 unique pathway conditions to increase limonene production in 24 hours from 0.2 to 4.5 mM (23 to 610 mg/L). Finally, to demonstrate the modularity of this pathway, we also synthesized the biofuel precursors pinene and bisabolene. We anticipate that iPROBE will accelerate design-build-test cycles for metabolic engineering, enabling data-driven multiplexed cell-free methods for testing large combinations of biosynthetic enzymes to inform cellular design.TOC FigureHighlightsApplied the iPROBE framework to build the nine-enzyme pathway to produce limoneneAssessed the impact of cofactors and 54 enzyme homologs on cell-free enzyme performanceIteratively optimized the cell-free production of limonene by exploring more than 580 unique reactionsExtended pathway to biofuel precursors pinene and bisabolene

Sign in / Sign up

Export Citation Format

Share Document