Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A / Unzueta, Ugutz; Vázquez, Felicitas; Accardi, Giulia; Mendoza, Rosa; Toledo Rubio, Verónica; Giuliani, Maria; Sannino, Filomena; Parrilli, Ermenegilda; Abasolo, Ibane; Schwartz, Simo; Tutino, MARIA LUISA; Villaverde, Antonio; Corchero, José L; Ferrer Miralles, Neus. - In: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. - ISSN 0175-7598. - 99:14(2015), pp. 5863-5874. [10.1007/s00253-014-6328-9]

Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A

PARRILLI, ERMENEGILDA;TUTINO, MARIA LUISA;
2015

Abstract

Obtaining high levels of pure proteins remains the main bottleneck of many scientific and biotechnological studies. Among all the available recombinant expression systems, Escherichia coli facilitates gene expression by its relative simplicity, inexpensive and fast cultivation, well-known genetics and the large number of tools available for its biotechnological application. However, recombinant expression in E. coli is not always a straightforward procedure and major obstacles are encountered when producing many eukaryotic proteins and especially membrane proteins, linked to missing posttranslational modifications, proteolysis and aggregation. In this context, many conventional and unconventional eukaryotic hosts are under exploration and development, but in some cases linked to complex culture media or processes. In this context, alternative bacterial systems able to overcome some of the limitations posed by E. coli keeping the simplicity of prokaryotic manipulation are currently emerging as convenient hosts for protein production. We have comparatively produced a "difficult-to-express" human protein, the lysosomal enzyme alpha-galactosidase A (hGLA) in E. coli and in the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC125 cells (P. haloplanktis TAC125). While in E. coli the production of active hGLA was unreachable due to proteolytic instability and/or protein misfolding, the expression of hGLA gene in P. haloplanktis TAC125 allows obtaining active enzyme. These results are discussed in the context of emerging bacterial systems for protein production that represent appealing alternatives to the regular use of E. coli and also of more complex eukaryotic systems.
2015
Strategies for the production of difficult-to-express full-length eukaryotic proteins using microbial cell factories: production of human alpha-galactosidase A / Unzueta, Ugutz; Vázquez, Felicitas; Accardi, Giulia; Mendoza, Rosa; Toledo Rubio, Verónica; Giuliani, Maria; Sannino, Filomena; Parrilli, Ermenegilda; Abasolo, Ibane; Schwartz, Simo; Tutino, MARIA LUISA; Villaverde, Antonio; Corchero, José L; Ferrer Miralles, Neus. - In: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY. - ISSN 0175-7598. - 99:14(2015), pp. 5863-5874. [10.1007/s00253-014-6328-9]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/612496
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