Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture.
Bioreactor
High cell density culture
Protein expression
Protein purification
Solubility tag
Journal
Microbial cell factories
ISSN: 1475-2859
Titre abrégé: Microb Cell Fact
Pays: England
ID NLM: 101139812
Informations de publication
Date de publication:
30 Jul 2021
30 Jul 2021
Historique:
received:
04
05
2021
accepted:
16
07
2021
entrez:
31
7
2021
pubmed:
1
8
2021
medline:
27
11
2021
Statut:
epublish
Résumé
The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for Escherichia coli. A fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures. The protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.
Sections du résumé
BACKGROUND
BACKGROUND
The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for Escherichia coli.
RESULTS
RESULTS
A fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures.
CONCLUSIONS
CONCLUSIONS
The protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.
Identifiants
pubmed: 34330289
doi: 10.1186/s12934-021-01638-8
pii: 10.1186/s12934-021-01638-8
pmc: PMC8325310
doi:
Substances chimiques
Adaptor Proteins, Signal Transducing
0
ITM2B protein, human
0
Molecular Chaperones
0
Recombinant Proteins
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
150Subventions
Organisme : Vetenskapsrådet
ID : 2020-02434
Organisme : Center for Innovative Medicine at Karolinska Institutet and Stockholm City Council
ID : 613/06
Organisme : Hjärnfonden
ID : FO2018-0312
Organisme : Stiftelsen Olle Engkvist Byggmästare
ID : 192-522
Organisme : Swedish Alzheimer foundation
ID : AF-836251
Organisme : Åhlén-stiftelsen
ID : mC9h18
Organisme : Petrus och Augusta Hedlunds Stiftelse
ID : M-2018-0998
Informations de copyright
© 2021. The Author(s).
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