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Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli
BACKGROUND: 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 express...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8922839/ https://www.ncbi.nlm.nih.gov/pubmed/35292023 http://dx.doi.org/10.1186/s12934-022-01766-9 |
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author | Balaban, Cecilia Lucía Suárez, Cristian Alejandro Boncompain, Carina Andrea Peressutti-Bacci, Natalia Ceccarelli, Eduardo Augusto Morbidoni, Héctor Ricardo |
author_facet | Balaban, Cecilia Lucía Suárez, Cristian Alejandro Boncompain, Carina Andrea Peressutti-Bacci, Natalia Ceccarelli, Eduardo Augusto Morbidoni, Héctor Ricardo |
author_sort | Balaban, Cecilia Lucía |
collection | PubMed |
description | BACKGROUND: 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. RESULTS: 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(2), 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. CONCLUSION: 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01766-9. |
format | Online Article Text |
id | pubmed-8922839 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-89228392022-03-22 Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli Balaban, Cecilia Lucía Suárez, Cristian Alejandro Boncompain, Carina Andrea Peressutti-Bacci, Natalia Ceccarelli, Eduardo Augusto Morbidoni, Héctor Ricardo Microb Cell Fact Research BACKGROUND: 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. RESULTS: 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(2), 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. CONCLUSION: 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01766-9. BioMed Central 2022-03-15 /pmc/articles/PMC8922839/ /pubmed/35292023 http://dx.doi.org/10.1186/s12934-022-01766-9 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Balaban, Cecilia Lucía Suárez, Cristian Alejandro Boncompain, Carina Andrea Peressutti-Bacci, Natalia Ceccarelli, Eduardo Augusto Morbidoni, Héctor Ricardo Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli |
title | Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli |
title_full | Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli |
title_fullStr | Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli |
title_full_unstemmed | Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli |
title_short | Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli |
title_sort | evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in escherichia coli |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8922839/ https://www.ncbi.nlm.nih.gov/pubmed/35292023 http://dx.doi.org/10.1186/s12934-022-01766-9 |
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