Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex
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
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.
cellulosome, assembly, affibody, affinity gene fusion, protein engineering, cellulose, bacterial receptor domain, binding domain, combinatorial library, degradation, expression, machines, affibody, chimeras, peptides, systems
IdentifiersURN: urn:nbn:se:kth:diva-23499DOI: 10.1016/j.jbiotec.2004.01.008ISI: 000221982700006ScopusID: 2-s2.0-16544388132OAI: oai:DiVA.org:kth-23499DiVA: diva2:342197
QC 201005252010-08-102010-08-102011-11-01Bibliographically approved