title: Fine-tuning sortase-mediated immobilization of protein layers on surfaces using sequential deprotection and coupling.
abstract: Increasing interest in protein immobilization on surfaces has heightened the need for techniques enabling layer-by-layer protein attachment. Here, we report a technique for controlling enzyme-mediated immobilization of layers of protein on the surface using a genetically encoded protecting group. An enterokinase-cleavable peptide sequence was inserted at the N-terminus of bifunctional fluorescent proteins containing Sortase A substrate recognition tags at both ends to control Sortase A-mediated protein immobilization on the surface layer-by-layer. Efficient, sequential immobilization of a second layer of protein using Sortase A required removal of the N-terminal protecting group, suggesting the method enables multilayer synthesis using cyclic deprotection and coupling steps. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:824-831, 2017.
authors.affiliation: Dept. of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, 37996.
authors.forename: Maryam
authors.initials: M
authors.lastname: Raeeszadeh-Sarmazdeh
authors.affiliation: Dept. of Bioengineering, University of Pennsylvania, Philadelphia, PA, 19114.
authors.forename: Ranganath
authors.initials: R
authors.lastname: Parthasarathy
authors.affiliation: Dept. of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, TN, 37996.
authors.forename: Eric T
authors.identifier: http://orcid.org/0000-0003-4095-5680
authors.initials: ET
authors.lastname: Boder
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D004765:Enteropeptidase / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D019881:Aminoacyltransferases; C120542:sortase A; D004765:Enteropeptidase; D003546:Cysteine Endopeptidases
keywords: protein immobilization; protein ligation; sortase A