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

Author:

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

Abstract

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.

Funder

Vetenskapsrådet

Center for Innovative Medicine at Karolinska Institutet and Stockholm City Council

Hjärnfonden

Stiftelsen Olle Engkvist Byggmästare

Swedish Alzheimer foundation

Åhlén-stiftelsen

Petrus och Augusta Hedlunds Stiftelse

Karolinska Institute

Publisher

Springer Science and Business Media LLC

Subject

Applied Microbiology and Biotechnology,Bioengineering,Biotechnology

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