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Site-specific and r...
Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex
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- Eklund, Malin (author)
- KTH,Bioteknologi
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- Sandström, Kristofer (author)
- KTH,Bioteknologi
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- Teeri, Tuula (author)
- KTH,Bioteknologi
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- Nygren, Per-Åke (author)
- KTH,Bioteknologi
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(creator_code:org_t)
- Elsevier BV, 2004
- 2004
- English.
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In: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 109:3, s. 277-286
- Related links:
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Subject headings
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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.
Subject headings
- NATURVETENSKAP -- Biologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences (hsv//eng)
Keyword
- cellulosome
- assembly
- affibody
- affinity gene fusion
- protein engineering
- cellulose
- bacterial receptor domain
- binding domain
- combinatorial library
- degradation
- expression
- machines
- affibody
- chimeras
- peptides
- systems
Publication and Content Type
- ref (subject category)
- art (subject category)
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