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

Cecilia Lucía Balaban, Cristian Alejandro Suárez, Carina Andrea Boncompain, Natalia Peressutti-Bacci, Eduardo Augusto Ceccarelli, Héctor Ricardo Morbidoni

Microbial cell factories 2022 Mar 15

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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, CaCl2, 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)6-tag location and lysis buffer additives (e.g. N-lauroylsarcosine), are sensible strategies to obtain higher levels of soluble S. aureus endolysins. © 2022. The Author(s).

Citation

Cecilia Lucía Balaban, Cristian Alejandro Suárez, Carina Andrea Boncompain, Natalia Peressutti-Bacci, Eduardo Augusto Ceccarelli, Héctor Ricardo Morbidoni. Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli. Microbial cell factories. 2022 Mar 15;21(1):40