Abstract
BackgroundThe 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.ResultsA 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.ConclusionsThe 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.
Highlights
The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases
Expression and purification of N-terminal domain (NT)*‐Bri2 BRICHOS in the bioreactor A typical yield of NT*-Bri2 BRICHOS is 80 mg/L when purified with IMAC after expression in a shake flask type cultivation, using Lysogeny Broth (LB) medium and inducing with 500 μM IPTG when the OD600 is below 1
Because we did not expect any significant improvement by optimizing the expression protocol using these complex media in terms of the cell density, we tested more sophisticated formulations, which included two semi-defined and one defined medium, originally described by da Silva, Korz, and Wyre (Culture #3, #4, and #5, respectively) [26,27,28]
Summary
The human Bri BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. Despite immense efforts from academia and the pharmaceutical industry, no disease-modifying treatment has been developed so far [2] This is a problem for the patients and their families and threatens to undermine the. Recombinant human (rh) Bri BRICHOS passes the blood-brain barrier in wild-type mice, making parenteral administration of rh Bri BRICHOS an interesting new potential treatment against AD [16, 17]. Such application of rh Bri BRICHOS would require industrial protein production and purification protocols that result in a homogenous product at a reasonable cost
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
