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Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.ORCID iD: 0000-0003-4214-6991
2004 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 109, no 3, 277-286 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2004. Vol. 109, no 3, 277-286 p.
Keyword [en]
cellulosome, assembly, affibody, affinity gene fusion, protein engineering, cellulose, bacterial receptor domain, binding domain, combinatorial library, degradation, expression, machines, affibody, chimeras, peptides, systems
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-23499DOI: 10.1016/j.jbiotec.2004.01.008ISI: 000221982700006Scopus ID: 2-s2.0-16544388132OAI: oai:DiVA.org:kth-23499DiVA: diva2:342197
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved

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Nygren, Per-Åke

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