Protein Immobilization On the Surface Using Sortase-Mediated Ligation

Protein immobilization on the surface using sortase-mediated ligation

Maryam Raeeszadeh-Sarmazdeh¹, Ranganath Parthasarathy², Eric T. Boder¹

¹ Department of Chemical and Biomolecular Engineering, University of Tennessee at Knoxville, Knoxville, TN 37996

²Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA 19104

The immobilization of proteins on surfaces is of great importance in molecular diagnostics and biosensors.  An enzymatic approach has distinct advantages compared to chemical methods since such immobilization can be achieved covalently and site-specifically in mild conditions compatible with protein structure and function. Sortase A is a transpeptidase that can sequence-specifically bind proteins or peptides to each other or to a surface.  Staphylococcus aureus sortase A recognizes a LPXTG sequence tag at the C-terminus of a protein and cleaves between threonine and glycine to yield an acyl-enzyme intermediate; this intermediate is resolved by nucleophilic attack from a gly_n (n>2) tag at the N-terminus of a second protein to link the two proteins through LPXT-G_n>2. By extension, a recombinant protein tagged with LPETG can be ligated in the desired orientation to a gly_n -functionalized surface. We have used sortase A to site-specifically bind recombinant eGFP (green fluorescent protein)-LPETG to a gold surface functionalized with triglycine using a cysteinyl linker. Fluorescence and atomic force microscopy show that protein immobilization is sortase A-mediated and tag-specific.  We also patterned eGFP on glass functionalized with Gly_n using a  Ni-NTA- PDMS stamp to capture his-tagged SrtA. Contact of the SrtA–loaded, patterned, stamp with the surface in the presence of eGFP-LPETG, produced patterned eGFP on the glass. As the next step toward creating well-defined “molecular print-boards,” we made layers of eGFP using a bifunctional eGFP with an LPETG sequence at the C-terminus and a GGG sequence immediately following a cleavable peptidic (genetically encoded) protecting group at the N-terminus.  Sequential deprotection-ligation steps enabled cyclic deposition of monomeric units in a manner analogous to other solid-phase synthesis applications, such as peptide or oligonucleotide synthesis.  Thus, layer-by-layer assembly of surface-attached, oriented molecular wires can be accomplished in a controlled manner.

mraeesza@utk.edu, rparthas@seas.upenn.edu, boder@utk.edu