Skip to main content
Research article - Peer-reviewed, 2021

Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture

Schmuck, Benjamin; Chen, Gefei; Pelcman, Josef; Kronqvist, Nina; Rising, Anna; Johansson, Jan

Abstract

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 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 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.

Keywords

Solubility tag; Bioreactor; High cell density culture; Protein expression; Protein purification

Published in

Microbial Cell Factories
2021, volume: 20, number: 1, article number: 150
Publisher: BMC

Authors' information

Karolinska Institute
Chen, Gefei
Karolinska Institutet
Pelcman, Josef
Karolinska Institutet
Kronqvist, Nina
Karolinska Institutet
Swedish University of Agricultural Sciences, Department of Anatomy, Physiology and Biochemistry (AFB)
Karolinska Institute
Johansson, Jan
Karolinska Institutet

UKÄ Subject classification

Microbiology

Publication Identifiers

DOI: https://doi.org/10.1186/s12934-021-01638-8

URI (permanent link to this page)

https://res.slu.se/id/publ/113292