Streamlining Escherichia Coli S30 Extract Preparation for Economical Cell-Free Protein Synthesis

2008 ◽  
Vol 21 (2) ◽  
pp. 460-465 ◽  
Author(s):  
David V. Liu ◽  
James F. Zawada ◽  
James R. Swartz
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Free Protein ◽  
S30 Extract ◽  
Cell Free Protein Synthesis

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

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 A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli

2019 ◽  
Vol 26 (12) ◽  
pp. 1743-1754.e9 ◽  
Author(s):  
Benjamin J. Des Soye ◽  
Vincent R. Gerbasi ◽  
Paul M. Thomas ◽  
Neil L. Kelleher ◽  
Michael C. Jewett
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
One Pot ◽  
Free Protein ◽  
Cell Free Protein Synthesis

scholarly journals Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Yong-Chan Kwon ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Host Cells ◽  
Persister Cells ◽  
Free Protein ◽  
E Coli ◽  
Dna And Rna ◽  
Rapid Production ◽  
Cell Free Protein Synthesis

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

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