Covalent attachment of proteins to solid supports and surfaces via Sortase-mediated ligation

• Published in: PloS one 2007-11, Vol.2 (11), p.e1164-e1164
• Main Authors: Chan, Lilyan, Cross, Hannah F, She, Joseph K, Cavalli, Gabriel, Martins, Hugo F P, Neylon, Cameron
• Format: Article
• Language: English
• Subjects: Amino acids; Base Sequence; Beads; Biochemistry; Biochemistry/Biocatalysis; Biochemistry/Biomacromolecule-Ligand Interactions; Biochemistry/Experimental Biophysical Methods; Biochemistry/Protein Chemistry; Biotechnology/Protein Chemistry and Proteomics; Catalysts; Chemistry; Crosslinking; Cysteine Endopeptidases - metabolism; Deoxyribonucleic acid; Devices; DNA; DNA Primers; E coli; Enzymes; Escherichia coli; Hydrolysis; Immobilization; Incorporation; Microscopy, Electron; Multiplexing; Peptides; Polymer crosslinking; Prostheses; Protein arrays; Protein Binding; Proteins; Proteins - metabolism; Sortase; Staphylococcus aureus
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0001164

There is growing interest in the attachment of proteins to solid supports for the development of supported catalysts, affinity matrices, and micro devices as well as for the development of planar and bead based protein arrays for multiplexed assays of protein concentration, interactions, and activity. A critical requirement for these applications is the generation of a stable linkage between the solid support and the immobilized, but still functional, protein. Solid supports including crosslinked polymer beads, beaded agarose, and planar glass surfaces, were modified to present an oligoglycine motif to solution. A range of proteins were ligated to the various surfaces using the Sortase A enzyme of S. aureus. Reactions were carried out in aqueous buffer conditions at room temperature for times between one and twelve hours. The Sortase A transpeptidase of S. aureus provides a general, robust, and gentle approach to the selective covalent immobilization of proteins on three very different solid supports. The proteins remain functional and accessible to solution. Sortase mediated ligation is therefore a straightforward methodology for the preparation of solid supported enzymes and bead based assays, as well as the modification of planar surfaces for microanalytical devices and protein arrays.