Abstract
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.
Highlights
Multidrug-resistant bacteria can be difficult to treat and are a serious threat to society [1]
Our group recently demonstrated that colicins, bacteriocins produced by Escherichia coli, can be produced using an E. coli-based cell-free protein synthesis (CFPS) system [9]
While our previous study showed that colicins E1, E2 and Ia were nearly completely soluble when produced in CFPS, we found that the majority of colicin M produced was insoluble and that only soluble colicin M (~5%) was active in killing K361 indicator cells [9]
Summary
Multidrug-resistant bacteria can be difficult to treat and are a serious threat to society [1]. A subgroup of antimicrobial peptides/proteins known as bacteriocins are considered to be viable alternatives to antibiotics because they exhibit high cell-killing activity against clinically important pathogens (both in vivo and in vitro), low oral toxicity to the host, as well as both broad- and narrow-spectrum qualities. Compared to cell-based protein production, cell-free protein synthesis (CFPS) provides several advantages for producing toxic proteins, as demonstrated in the cases of onconase (RNase) [6], pierisin-1b [7], cecropin P1 [8], and colicins [9], which are deleterious to the host cells when overproduced. We reported that colicins E1 and E2 are very effective in killing antibiotic-tolerant persister cells [9] Some colicins such as colicin M exhibited low solubility and poor cell-killing activity when produced in CFPS. Further improvement of colicin production and bioactivity is required for optimal cell-free colicin production
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