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

Schmuck, Benjamin and Chen, Gefei and Pelcman, Josef and Kronqvist, Nina and Rising, Anna and Johansson, Jan (2021). Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture. Microbial Cell Factories. 20 , 150
[Research article]

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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.

Authors/Creators:Schmuck, Benjamin and Chen, Gefei and Pelcman, Josef and Kronqvist, Nina and Rising, Anna and Johansson, Jan
Title:Expression of the human molecular chaperone domain Bri2 BRICHOS on a gram per liter scale with an E. coli fed-batch culture
Series Name/Journal:Microbial Cell Factories
Year of publishing :2021
Volume:20
Article number:150
Number of Pages:12
Publisher:BMC
ISSN:1475-2859
Language:English
Publication Type:Research article
Article category:Scientific peer reviewed
Version:Published version
Copyright:Creative Commons: Attribution 4.0
Full Text Status:Public
Subjects:(A) Swedish standard research categories 2011 > 1 Natural sciences > 106 Biological Sciences (Medical to be 3 and Agricultural to be 4) > Microbiology (Microbiology in the medical area to be 30109)
Keywords:Solubility tag, Bioreactor, High cell density culture, Protein expression, Protein purification
URN:NBN:urn:nbn:se:slu:epsilon-p-113292
Permanent URL:
http://urn.kb.se/resolve?urn=urn:nbn:se:slu:epsilon-p-113292
Additional ID:
Type of IDID
DOI10.1186/s12934-021-01638-8
Web of Science (WoS)000679729600001
ID Code:25149
Faculty:VH - Faculty of Veterinary Medicine and Animal Science
Department:(VH) > Dept. of Anatomy, Physiology and Biochemistry
Deposited By: SLUpub Connector
Deposited On:02 Sep 2021 09:25
Metadata Last Modified:02 Sep 2021 09:31

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