Covalent Attachment of Proteins to Solid Supports and Surfaces via Sortase-Mediated Ligation

Abstract

BackgroundThere 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.MethodologySolid 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.ConclusionsThe 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.

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

“…Residues in strands ␤4 and ␤7 form the floor of the groove, whereas the walls are formed by surface loops that connect strand ␤6 to strand ␤7 (␤6/␤7 loop), strand ␤7 to strand ␤8 (␤7/␤8 loop), strand ␤3 to strand ␤4 (␤3/␤4 loop), and strand ␤2 to helix H1 (␤2/H1 loop). The leucine residue of the signal rests against the ␤6/␤7 loop, where residues Val 166 -Leu 169 adopt a 3 10 helix that only forms when the substrate is bound (Fig. 2c).…”

Sortase‐Mediated Ligation: A Gift from Gram‐Positive Bacteria to Protein Engineering

“…[44] Although peptides carrying only a single N-terminal glycine have been shown to participate in the sortase-catalyzed transpeptidation, maximum reaction efficiency is generally obtained with substrates in which two or more glycines are incorporated. [45][46][47] On the other hand, and more significantly, SrtA is promiscuous with respect to the flanking sequences. As we can see in the next section, various molecules can be used as substrates for SrtA by fusing them to the LPXTG and a-Gly n tags.…”

“…As we can see in the next section, various molecules can be used as substrates for SrtA by fusing them to the LPXTG and a-Gly n tags. [46][47][48][49][50][51][52][53][54][55][56][57][58][59] As another interesting feature, SrtA is also capable of using several primary amine derivatives such as hydroxylamine [38] and alkylamines [49,58] as a-Gly n surrogates, though these non-peptidic substrates appear to be less efficient than oligoglycine derivatives.…”

“…[47] When Tus-LPETG-His 6 , a sequence-specific DNA binding protein, was A C H T U N G T R E N N U N G immobilized on a-Gly 4 -conjugated methacrylate beads, it retained almost full binding affinity and specificity toward its cognate TerB DNA ligand. Very recently, Proft and co-workers also used SML to fabricate a protein sensor chip on which fibronectin-binding protein (Fba) was covalently attached.…”

Reprogramming the specificity of sortase enzymes

“…1A). The small size of the sorting motif and the synthetic accessibility of Gly-GlyGly-linked substrates have led to the use of SrtA in a many applications including the synthesis of protein-protein (4), protein-nucleic acid (6), protein-lipid (7), and protein-surface (8) conjugates.…”