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

Autores
Balaban, Cecilia Lucía; Suárez, Cristian Alejandro; Boncompain, Carina Andrea; Peressutti Bacci, Natalia; Ceccarelli, Eduardo Augusto; Morbidoni, Héctor Ricardo
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
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, 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. 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.
Fil: Balaban, Cecilia Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina
Fil: Suárez, Cristian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina
Fil: Boncompain, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina
Fil: Peressutti Bacci, Natalia. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina
Fil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina
Materia
BACTERIOPHAGES
CHAPERONES
ENDOLYSINS
RECOMBINANT EXPRESSION
SOLUBILITY
STAPHYLOCOCCUS AUREUS
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/216315
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/216315
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coliBalaban, Cecilia LucíaSuárez, Cristian AlejandroBoncompain, Carina AndreaPeressutti Bacci, NataliaCeccarelli, Eduardo AugustoMorbidoni, Héctor RicardoBACTERIOPHAGESCHAPERONESENDOLYSINSRECOMBINANT EXPRESSIONSOLUBILITYSTAPHYLOCOCCUS AUREUShttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Background: 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, 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. 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.Fil: Balaban, Cecilia Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Suárez, Cristian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: Boncompain, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaFil: Peressutti Bacci, Natalia. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; ArgentinaFil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; ArgentinaBioMed Central2022-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/216315Balaban, Cecilia Lucía; Suárez, Cristian Alejandro; Boncompain, Carina Andrea; Peressutti Bacci, Natalia; Ceccarelli, Eduardo Augusto; et al.; Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli; BioMed Central; Microbial Cell Factories; 21; 1; 3-2022; 1-191475-2859CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-022-01766-9#citeasinfo:eu-repo/semantics/altIdentifier/doi/10.1186/s12934-022-01766-9info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:13:46Zoai:ri.conicet.gov.ar:11336/216315instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:13:46.84CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv 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
spellingShingle Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli
Balaban, Cecilia Lucía
BACTERIOPHAGES
CHAPERONES
ENDOLYSINS
RECOMBINANT EXPRESSION
SOLUBILITY
STAPHYLOCOCCUS AUREUS
title_short 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_sort Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli
dc.creator.none.fl_str_mv Balaban, Cecilia Lucía
Suárez, Cristian Alejandro
Boncompain, Carina Andrea
Peressutti Bacci, Natalia
Ceccarelli, Eduardo Augusto
Morbidoni, Héctor Ricardo
author Balaban, Cecilia Lucía
author_facet Balaban, Cecilia Lucía
Suárez, Cristian Alejandro
Boncompain, Carina Andrea
Peressutti Bacci, Natalia
Ceccarelli, Eduardo Augusto
Morbidoni, Héctor Ricardo
author_role author
author2 Suárez, Cristian Alejandro
Boncompain, Carina Andrea
Peressutti Bacci, Natalia
Ceccarelli, Eduardo Augusto
Morbidoni, Héctor Ricardo
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv BACTERIOPHAGES
CHAPERONES
ENDOLYSINS
RECOMBINANT EXPRESSION
SOLUBILITY
STAPHYLOCOCCUS AUREUS
topic BACTERIOPHAGES
CHAPERONES
ENDOLYSINS
RECOMBINANT EXPRESSION
SOLUBILITY
STAPHYLOCOCCUS AUREUS
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv 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, 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. 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.
Fil: Balaban, Cecilia Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina
Fil: Suárez, Cristian Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina
Fil: Boncompain, Carina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina
Fil: Peressutti Bacci, Natalia. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina
Fil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina
Fil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Laboratorio de Microbiología Molecular; Argentina
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, 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. 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.
publishDate 2022
dc.date.none.fl_str_mv 2022-03
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/216315
Balaban, Cecilia Lucía; Suárez, Cristian Alejandro; Boncompain, Carina Andrea; Peressutti Bacci, Natalia; Ceccarelli, Eduardo Augusto; et al.; Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli; BioMed Central; Microbial Cell Factories; 21; 1; 3-2022; 1-19
1475-2859
CONICET Digital
CONICET
url http://hdl.handle.net/11336/216315
identifier_str_mv Balaban, Cecilia Lucía; Suárez, Cristian Alejandro; Boncompain, Carina Andrea; Peressutti Bacci, Natalia; Ceccarelli, Eduardo Augusto; et al.; Evaluation of factors influencing expression and extraction of recombinant bacteriophage endolysins in Escherichia coli; BioMed Central; Microbial Cell Factories; 21; 1; 3-2022; 1-19
1475-2859
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
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info:eu-repo/semantics/altIdentifier/doi/10.1186/s12934-022-01766-9
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
application/pdf
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dc.publisher.none.fl_str_mv BioMed Central
publisher.none.fl_str_mv BioMed Central
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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