Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex

Malin Eklund, K Sandstrom, TT Teeri, PA Nygren*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

Abstract

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. (C) 2004 Elsevier B.V. All rights reserved.

Original languageEnglish
Pages (from-to)277-286
Number of pages10
JournalJournal of Biotechnology
Volume109
Issue number3
DOIs
Publication statusPublished - 29 Apr 2004
MoE publication typeA1 Journal article-refereed

Keywords

  • cellulosome
  • assembly
  • affibody
  • affinity gene fusion
  • protein engineering
  • cellulose
  • BACTERIAL RECEPTOR DOMAIN
  • BINDING DOMAIN
  • COMBINATORIAL LIBRARY
  • DEGRADATION
  • EXPRESSION
  • MACHINES
  • AFFIBODY
  • CHIMERAS
  • PEPTIDES
  • SYSTEMS

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