title: Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases.
abstract: We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.
authors.affiliation: Department of Chemical and Biomolecular Engineering and Institute for Biomedical Engineering, University of Tennessee at Knoxville, Knoxville, TN 37996, United States.
authors.forename: Maryam
authors.initials: M
authors.lastname: Raeeszadeh-Sarmazdeh
authors.affiliation: Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19114, United States.
authors.forename: Ranganath
authors.initials: R
authors.lastname: Parthasarathy
authors.affiliation: Department of Chemical and Biomolecular Engineering and Institute for Biomedical Engineering, University of Tennessee at Knoxville, Knoxville, TN 37996, United States. Electronic address: boder@utk.edu.
authors.forename: Eric T
authors.initials: ET
authors.lastname: Boder
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D036103:Nanotechnology / Q000295:instrumentation / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology; D013297:Streptococcus pyogenes / Q000737:chemistry / Q000201:enzymology; D013499:Surface Properties; D000097573:Red Fluorescent Protein
chemical_list: D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases; D006046:Gold; D049452:Green Fluorescent Proteins; D055767:Immobilized Proteins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C120542:sortase A
keywords: Orthogonal sortases; Protein 3D assembly; Protein immobilization on gold; Site-specific protein ligation; Sortase A