Sequence-, orientation-, and site-specific ligation of proteins to surfaces presents considerable challenges. We have shown that the transpeptidase, Sortase A, from the Gram-positive bacterium Staphylococcus aureus is a versatile enzyme that can accomplish sequence-specific ligation of proteins to surfaces as well as to soluble species. The ligation requires a carboxy-terminal pentapeptide tag, ---LPXTG on one reactant (R1-LPXTG) and an amino-terminal (Gly)n , n≥2, on the other (GGG-R2). A protein tagged with -LPETG can thus be ligated to a surface with an amino-GGG resulting in orientation-specific presentation of the protein with a tether at its carboxy terminus.

Site-specific modification of surfaces with high spatial resolution has been accomplished using either dip-pen lithography or, more recently, with topographically etched stamps, wherein tethering an enzyme to the stamp greatly increases spatial resolution. We have used Sortase A to spatially pattern protein onto surfaces by contact printing. Sortase A was immobilized on a micro-patterned PDMS stamp and used to couple LPETG-tagged enhanced green fluorescent protein (eGFP) to Gly3-modified glass surfaces. The stamped protein is covalently coupled to the glass and demonstrates high fidelity replication of the spatial pattern of the PDMS stamp. Since one of the benefits of microcontact printing is the possibility of cross-patterning using multiple enzymes with unique target sites, we have also conducted preliminary work to characterize Sortase A from Corynebacterium diphtheriae. In vivo, this enzyme replaces the Glyn substrate of the S. aureus homolog with a lysine side chain contained within a conserved stretch of amino acids (the "pilin box"). Solution data suggest functional overlap between the two homologs in terms of recognition sites. The rate of transition state hydrolysis is faster with the C. diptheriae Sortase A relative to the S. aureus enzyme.

Sortase-mediated ligation has the advantages of using gentle conditions, requiring recognition sequences that are only 3-5 amino acids long, and specifically coupling proteins from a complex mixture; these features render the method versatile and compatible with the functions of many proteins. We anticipate that Sortase-catalyzed microcontact printing will be a powerful tool with which to modify surface physical properties on very small scales in addition to the obvious applications in protein array construction.