Benjamin Schmuck1,2, Gefei Chen3, Josef Pelcman3, Nina Kronqvist3, Anna Rising3,4, Jan Johansson3. 1. Department of Biosciences and Nutrition, Karolinska Institutet, Neo, 141 86, Huddinge, Sweden. benjamin.schmuck@ki.se. 2. Department of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. benjamin.schmuck@ki.se. 3. Department of Biosciences and Nutrition, Karolinska Institutet, Neo, 141 86, Huddinge, Sweden. 4. Department of Anatomy, Physiology, and Biochemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden.
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.
BACKGROUND: The humanBri2 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: Hanna Willander; Glareh Askarieh; Michael Landreh; Per Westermark; Kerstin Nordling; Henrik Keränen; Erik Hermansson; Aaron Hamvas; Lawrence M Nogee; Tomas Bergman; Alejandra Saenz; Cristina Casals; Johan Åqvistg; Hans Jörnvall; Helena Berglund; Jenny Presto; Stefan D Knight; Jan Johansson Journal: Proc Natl Acad Sci U S A Date: 2012-02-02 Impact factor: 11.205
Authors: Hanna Willander; Jenny Presto; Glareh Askarieh; Henrik Biverstål; Birgitta Frohm; Stefan D Knight; Jan Johansson; Sara Linse Journal: J Biol Chem Date: 2012-07-16 Impact factor: 5.157
Authors: Bengt Winblad; Philippe Amouyel; Sandrine Andrieu; Clive Ballard; Carol Brayne; Henry Brodaty; Angel Cedazo-Minguez; Bruno Dubois; David Edvardsson; Howard Feldman; Laura Fratiglioni; Giovanni B Frisoni; Serge Gauthier; Jean Georges; Caroline Graff; Khalid Iqbal; Frank Jessen; Gunilla Johansson; Linus Jönsson; Miia Kivipelto; Martin Knapp; Francesca Mangialasche; René Melis; Agneta Nordberg; Marcel Olde Rikkert; Chengxuan Qiu; Thomas P Sakmar; Philip Scheltens; Lon S Schneider; Reisa Sperling; Lars O Tjernberg; Gunhild Waldemar; Anders Wimo; Henrik Zetterberg Journal: Lancet Neurol Date: 2016-04 Impact factor: 44.182
Authors: M Ankarcrona; B Winblad; C Monteiro; C Fearns; E T Powers; J Johansson; G T Westermark; J Presto; B-G Ericzon; J W Kelly Journal: J Intern Med Date: 2016-05-10 Impact factor: 8.989
Authors: R Vidal; T Revesz; A Rostagno; E Kim; J L Holton; T Bek; M Bojsen-Møller; H Braendgaard; G Plant; J Ghiso; B Frangione Journal: Proc Natl Acad Sci U S A Date: 2000-04-25 Impact factor: 11.205
Authors: Charlotte Nerelius; Magnus Gustafsson; Kerstin Nordling; Annika Larsson; Jan Johansson Journal: Biochemistry Date: 2009-05-05 Impact factor: 3.162
Authors: Marie E Oskarsson; Erik Hermansson; Ye Wang; Nils Welsh; Jenny Presto; Jan Johansson; Gunilla T Westermark Journal: Proc Natl Acad Sci U S A Date: 2018-03-05 Impact factor: 11.205
Authors: Anna Katharina Schellhaus; Shanshan Xu; Maria E Gierisch; Julia Vornberger; Jan Johansson; Nico P Dantuma Journal: Commun Biol Date: 2022-05-26