Site-specific and reversible anchoring of active proteins onto cellulose using a cellulosome-like complex.
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 cellulose-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 (CBM1Cel6A) 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-CBM1Cel6A fusion protein had been previously applied. This showed that the SPA-CBM1Cel6A 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.
Eklund
Malin
M
Department of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden.
Sandström
Kristofer
K
Teeri
Tuula T
TT
Nygren
Per-Ake
PA
D002482:Cellulose / Q000737:chemistry*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics; D044603:Cellulosomes; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics; D005670:Fusarium / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics; D046912:Multiprotein Complexes / Q000737:chemistry*; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000235:genetics; D014242:Trichoderma / Q000235:genetics
D004800:Enzymes, Immobilized; D046912:Multiprotein Complexes; D019151:Peptide Library; D010455:Peptides; D013205:Staphylococcal Protein A; D049452:Green Fluorescent Proteins; D002482:Cellulose; D008049:Lipase; D043366:Cellulose 1,4-beta-Cellobiosidase