Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli

Endolysins are peptidoglycan hydrolases with promising use as environment-friendly antibacterials mainly when used topically. However, in general, endolysin expression is hampered by its low solubility. Thus, a critical point in endolysin industrial production is optimizing their expression, includi...

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Published in:	Microbial cell factories Vol. 21; no. 1; pp. 40 - 14
Main Authors:	Balaban, Cecilia Lucía, Suárez, Cristian Alejandro, Boncompain, Carina Andrea, Peressutti-Bacci, Natalia, Ceccarelli, Eduardo Augusto, Morbidoni, Héctor Ricardo
Format:	Journal Article
Language:	English
Published:	England BioMed Central Ltd 15-03-2022 BioMed Central BMC
Subjects:	Analysis Bacteriophages Bacteriophages - genetics Chaperones Drug resistance in microorganisms Endolysins Endopeptidases - genetics Endopeptidases - metabolism Escherichia coli Escherichia coli - genetics Escherichia coli - metabolism Gene expression Genetic aspects Recombinant expression Solubility Staphylococcus aureus Staphylococcus aureus - metabolism Argentina Bacteriophages Recombinant expression Endolysins Chaperones Staphylococcus aureus Solubility
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Summary:	Endolysins are peptidoglycan hydrolases with promising use as environment-friendly antibacterials mainly when used topically. However, in general, endolysin expression is hampered by its low solubility. Thus, a critical point in endolysin industrial production is optimizing their expression, including improvement of solubility and recovery from cell extracts. We report the expression of two endolysins encoded in the genome of phages infecting Staphylococcus aureus. Expression was optimized through changes in the concentration of the inducer and growth temperature during the expression. Usually, only 30-40% of the total endolysin was recovered in the soluble fraction. Co-expression of molecular chaperones (DnaK, GroEL) or N-term fusion tags endowed with increased solubility (DsbC, Trx, Sumo) failed to improve that yield substantially. Inclusion of osmolytes (NaCl, CaCl , mannitol, glycine betaine, glycerol and trehalose) or tensioactives (Triton X-100, Tween 20, Nonidet P-40, CHAPS, N-lauroylsarcosine) in the cell disruption system (in the absence of any molecular chaperone) gave meager improvements excepted by N-lauroylsarcosine which increased recovery to 54% of the total endolysin content. This is the first attempt to systematically analyze methods for increasing yields of recombinant endolysins. We herein show that neither solubility tags nor molecular chaperones co-expression are effective to that end, while induction temperature, (His) -tag location and lysis buffer additives (e.g. N-lauroylsarcosine), are sensible strategies to obtain higher levels of soluble S. aureus endolysins.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1475-2859 1475-2859
DOI:	10.1186/s12934-022-01766-9