Analysis of the expression of cloned genes using an Escherichia coli cell-free system

1982 ◽  
Vol 60 (12) ◽  
pp. 1095-1100 ◽  
Author(s):  
Yew Phew See ◽  
Bernard R. Glick
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Restriction Enzyme ◽  
Coli Cell ◽  
Free System ◽  
Low Background ◽  
Cell Free System ◽  
Endogenous Protein ◽  
Circular Dnas

An Escherichia coli coupled transcription–translation cell-free system, which is efficient in the synthesis of proteins directed by exogenously added DNA, is described. These cell-free extracts direct protein synthesis against a low background of endogenous protein synthesis providing a means for analyzing the expression of isolated genes. This is especially important when using restriction enzyme-linearized DNAs which are less efficient templates than circular DNAs. This cell-free system has been used to study the expression of the proteins coded by plasmids pBR322 and pBL101.

Synthesis of yeast histone 3 in an Escherichia coli cell-free system

1983 ◽  
Vol 168 (3) ◽  
pp. 489-503 ◽  
Author(s):  
Rafael P. Mellado ◽  
Kenneth Murray ◽  
P. Chambon
Keyword(s):  
Escherichia Coli ◽  
Coli Cell ◽  
Free System ◽  
Cell Free System ◽  
Histone 3

scholarly journals Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins

2019 ◽  
Vol 20 (3) ◽  
pp. 492 ◽  
Author(s):  
Jiro Adachi ◽  
Kazushige Katsura ◽  
Eiko Seki ◽  
Chie Takemoto ◽  
Mikako Shirouzu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Protein Synthesis ◽  
Trna Synthetase ◽  
Translation Efficiency ◽  
Free System ◽  
Free Protein ◽  
Cell Free System ◽  
Natural Amino Acids ◽  
Cell Free Protein Synthesis

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

High-level soluble expression of one model olfactory receptor (ODR-10) in Escherichia coli cell-free system

2013 ◽  
Vol 30 (3) ◽  
pp. 893-901 ◽  
Author(s):  
Xu Zhang ◽  
Jiayuan Sheng ◽  
Lei Huang ◽  
Liping Du ◽  
Jin Cai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Olfactory Receptor ◽  
Soluble Expression ◽  
Coli Cell ◽  
Free System ◽  
Cell Free System ◽  
High Level
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