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Milligram scale expression, refolding, and purification of Bombyx mori cocoonase using a recombinant E. coli system
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Department of Bio-Engineering, Royal University of Phnom Penh, Phnom Penh, Cambodia.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.ORCID-id: 0000-0002-5636-2567
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.ORCID-id: 0000-0002-9098-7974
2021 (Engelska)Ingår i: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 186, artikel-id 105919Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Silk is one of the most versatile biomaterials with signature properties of outstanding mechanical strength and flexibility. A potential avenue for developing more environmentally friendly silk production is to make use of the silk moth (Bombyx mori) cocoonase, this will at the same time increase the possibility for using the byproduct, sericin, as a raw material for other applications. Cocoonase is a serine protease utilized by the silk moth to soften the cocoon to enable its escape after completed metamorphosis. Cocoonase selectively degrades the glue protein of the cocoon, sericin, without affecting the silk-fiber made of the protein fibroin. Cocoonase can be recombinantly produced in E. coli, however, it is exclusively found as insoluble inclusion bodies. To solve this problem and to be able to utilize the benefits associated with an E. coli based expression system, we have developed a protocol that enables the production of soluble and functional protease in the milligram/liter scale. The core of the protocol is refolding of the protein in a buffer with a redox potential that is optimized for formation of native and intramolecular di-sulfide bridges. The redox potential was balanced with defined concentrations of reduced and oxidized glutathione. This E. coli based production protocol will, in addition to structure determination, also enable modification of cocoonase both in terms of catalytic function and stability. These factors will be valuable components in the development of alternate silk production methodology.

Ort, förlag, år, upplaga, sidor
Elsevier, 2021. Vol. 186, artikel-id 105919
Nyckelord [en]
Cocoonase, Escherichia coli, Refolding, Serine protease, Silk moth (Bombyx mori)
Nationell ämneskategori
Biokemi och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:umu:diva-184200DOI: 10.1016/j.pep.2021.105919ISI: 000671874800003Scopus ID: 2-s2.0-85106960638OAI: oai:DiVA.org:umu-184200DiVA, id: diva2:1565750
Forskningsfinansiär
Vetenskapsrådet, 2017–04203Kempestiftelserna, JCK-1417Tillgänglig från: 2021-06-14 Skapad: 2021-06-14 Senast uppdaterad: 2023-09-05Bibliografiskt granskad

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Phoeurk, ChanrithUl Mushtaq, AmeeqRogne, PerWolf-Watz, Magnus

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Protein Expression and Purification
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