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Site-specific and r...
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Eklund, MalinKTH,Bioteknologi
(författare)
Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex
- Artikel/kapitelEngelska2004
Förlag, utgivningsår, omfång ...
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Elsevier BV,2004
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printrdacarrier
Nummerbeteckningar
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LIBRIS-ID:oai:DiVA.org:kth-23499
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https://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-23499URI
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https://doi.org/10.1016/j.jbiotec.2004.01.008DOI
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Språk:engelska
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Sammanfattning på:engelska
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Ämneskategori:ref swepub-contenttype
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Ämneskategori:art swepub-publicationtype
Anmärkningar
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QC 20100525
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Protein engineering strategies facilitating controlled and spontaneous assembly of macromolecular complexes are of great interest for the design of artificial multi-enzyme systems of pre-defined composition. Here we have combined affinity proteins from different sources to achieve specific and reversible anchoring of affinity domain-tagged reporter proteins to a cell ulose-anchored fusion protein. The design principle mimics the architecture of macromolecular cellulosome complexes produced by some cellulolytic microbes. A fusion protein between a cellulose-binding module (CBM1(Cel6A)) of the Trichoderma reesei cellobiohydrolase Cel6A and a five-domain staphylococcal protein A (SPA) was constructed to serve as platform for docking of easily detectable reporter proteins onto cellulose surfaces. In turn, the reporter proteins were produced as fusions to two copies of a SPA-binding affinity protein (an affibody denoted Z(SPA-1)), selected from a phage display library constructed by combinatorial protein engineering. In a series of experiments, involving repeated washing and low pH elution, affinity-tagged Enhanced Green Fluorescent Protein (EGFP) and Fusarium solani pisi lipase cutinase reporter proteins were both found to be specifically directed from solution to the same region of a cellulose filter paper where SPA-CBM1(Cel6A) fusion protein had been previously applied. This showed that the SPA-CBM1(Cel6A) fusion protein had been stably anchored to the cellulose surface without loss of binding capacity and that the interaction between SPA and the Z(SPA-1) affibody domains was selective. The generality of this biospecificity-driven system for assembly applications is discussed.
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Biuppslag (personer, institutioner, konferenser, titlar ...)
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Sandström, KristoferKTH,Bioteknologi(Swepub:kth)u1qc3vv1
(författare)
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Teeri, TuulaKTH,Bioteknologi(Swepub:kth)u1vcejiv
(författare)
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Nygren, Per-ÅkeKTH,Bioteknologi(Swepub:kth)u1zhverl
(författare)
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KTHBioteknologi
(creator_code:org_t)
Sammanhörande titlar
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Ingår i:Journal of Biotechnology: Elsevier BV109:3, s. 277-2860168-16561873-4863
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