title: Synthesis and characterization of bioconjugates of S-layer proteins.
abstract: The self-assembling proteins that form crystalline surface layers (S-layers) on many microbial species have found numerous applications due to their nanostructured nature. To devise a new method to construct surface displays that exploit S-layer self-assembly activity and nanostructural properties, we have constructed polymer bioconjugates of S-layer proteins. The conjugates formed are similar in function to the monomer alkanethiols that form self-assembled monolayers (SAMs) on gold surfaces. However, the self-assembly is driven by the protein "headgroup" that positions polymer-tethered endgroups on a surface. This paper examines the integration of protein purification, conjugation, and surface assembly that has led to the development of this new method for the formation of nanostructured surfaces. Purified S-layer proteins from Lactobacillus brevis were conjugated with small molecule probes and polymers using amine-based reactions. To keep multiple labeling of protein amine groups to acceptable levels, the conjugations were performed at pH 6.5, allowing for limited yields (24-39%) as determined by mass spectrometry and SDS-polyacrylamide gel electrophoresis. As the presence of high levels of unlabeled S-layer proteins is undesired, we have developed a protocol for further purification that employs monomeric avidin affinity chromatography. The surface self-assembly of the polymer bioconjugates onto amine-terminated microspheres was studied using epi-fluorescence, confocal, and scanning electron microscopy. The surfaces obtained exhibited homogeneous distributions of tethered molecules. Also, in cases where the modular assembly of two distinct types of tethered endgroups was accomplished, there was no evidence for phase separation in the surfaces. The modular assembly method will provide a potential route to controlling surface display density as the starting assembly conditions guide displayed endgroup concentrations in mixed molecular monolayers.
authors.affiliation: Department of Chemical Engineering, The City College of the City University of New York, New York, New York 10031, USA.
authors.forename: Parthasarathy
authors.initials: P
authors.lastname: Sampathkumar
authors.forename: M Lane
authors.initials: ML
authors.lastname: Gilchrist
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001704:Biopolymers / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003720:Densitometry; D007778:Lactobacillus / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018613:Microscopy, Confocal; D008863:Microspheres; D036103:Nanotechnology; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D001704:Biopolymers; D008562:Membrane Glycoproteins; C489049:regular S-layer protein, bacteria; D011092:Polyethylene Glycols