A Protocol for Expression and Purication of Cyclic Nucleotide–Free Protein in Escherichia coli

2015 ◽  
pp. 208-219
Keyword(s):  
Escherichia Coli ◽  
Cyclic Nucleotide ◽  
Free Protein

scholarly journals Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins

2019 ◽  
Vol 2 (2) ◽  
pp. 28 ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Cell Killing ◽  
Great Promise ◽  
Immunity Protein ◽  
Free Protein ◽  
Killing Activity ◽  
Colicin E1 ◽  
Colicin E3 ◽  
Cell Free Protein Synthesis

Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are effective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we confirmed our previous finding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our findings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform.

scholarly journals A Highly Efficient Cell-Free Protein Synthesis System from Escherichia coli

1996 ◽  
Vol 239 (3) ◽  
pp. 881-886 ◽  
Author(s):  
Dong-Myung Kim ◽  
Takanori Kigawa ◽  
Cha-Yong Choi ◽  
Shigeyuki Yokoyama
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Synthesis System ◽  
Free Protein ◽  
Highly Efficient ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

scholarly journals RNase I regulates Escherichia coli 2′,3′-cyclic nucleotide monophosphate levels and biofilm formation

2018 ◽  
Vol 475 (8) ◽  
pp. 1491-1506 ◽  
Author(s):  
Benjamin M. Fontaine ◽  
Kevin S. Martin ◽  
Jennifer M. Garcia-Rodriguez ◽  
Claire Jung ◽  
Laura Briggs ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Cyclic Nucleotide ◽  
Biofilm Formation ◽  
Physiological Role ◽  
Rna Degradation ◽  
Bacterial Biofilm ◽  
Biological Functions ◽  
Salvage Pathway ◽  
E Coli

Regulation of nucleotide and nucleoside concentrations is critical for faithful DNA replication, transcription, and translation in all organisms, and has been linked to bacterial biofilm formation. Unusual 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) recently were quantified in mammalian systems, and previous reports have linked these nucleotides to cellular stress and damage in eukaryotes, suggesting an intriguing connection with nucleotide/nucleoside pools and/or cyclic nucleotide signaling. This work reports the first quantification of 2′,3′-cNMPs in Escherichia coli and demonstrates that 2′,3′-cNMP levels in E. coli are generated specifically from RNase I-catalyzed RNA degradation, presumably as part of a previously unidentified nucleotide salvage pathway. Furthermore, RNase I and 2′,3′-cNMP levels are demonstrated to play an important role in controlling biofilm formation. This work identifies a physiological role for cytoplasmic RNase I and constitutes the first progress toward elucidating the biological functions of bacterial 2′,3′-cNMPs.

Sign in / Sign up

Export Citation Format

Share Document