Synthesis and characterization of S -layer protein bioconjugates: A route to biologically-based self -assembled monolayers.
Item
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Title
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Synthesis and characterization of S -layer protein bioconjugates: A route to biologically-based self -assembled monolayers.
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Identifier
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AAI3144136
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identifier
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3144136
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Creator
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Sampathkumar, Parthasarathy.
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Contributor
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Adviser: M. Lane Gilchrist
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Date
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2004
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Language
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English
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Publisher
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City University of New York.
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Subject
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Engineering, Chemical | Chemistry, Biochemistry
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Abstract
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Numerous bacterial species possess nanostructured monolayers of self-assembled proteins on their surfaces called the crystalline surface layer (S-layer) that efficiently display molecules. We utilize these protein-based molecular display systems to develop novel, multicomponent bioactive nanostructured surfaces. Essentially, S-layer proteins were conjugated with specific tethered molecules and subsequently self-assembled on surfaces using combinations of bioconjugated protein monomer "modules". The bioconjugate modules are similar in function to the alkanethiols and silanes that form self-assembled monolayers (SAMs) on respectively gold and silicon surfaces. The S-layer proteins in comparison, serve as the "headgroups" or molecular carriers and the driving force for the formation of SAMs positioning the tethered "endgroups" on the surface. The inherent nanostructure of the S-layer crystal lattice dictates the endgroup orientation and spacing.;We have isolated and purified the S-layer proteins from microbial cultures of Lactobacillus brevis and Bacillus sphaericus. S-layer protein bioconjugate modules have been built with small molecule probes and polyethylene glycol (PEG) tethered ligands using amine-based reactions. The conjugations were performed at pH 6.5 to limit multiple labeling of protein amine groups. The modules have been characterized using HPLC, mass spectrometry and SDS-PAGE. Yields of singly N-terminus labeled proteins were 24--39%. To reduce the percentage of unlabeled S-layer proteins and further purify these modules, we have developed a protocol employing monomeric avidin affinity chromatography. We have assembled our bioconjugate modules onto polymer microspheres, silicon chips, positively charged glass slides and cationic liposomes. The distribution of modules in the assembled surfaces was followed using fluorescence spectroscopy and confocal microscopy. The surface-assembled structures were also examined with TEM, SEM, FT-IR and AFM. The surfaces obtained exhibited homogeneous distributions of tethered molecules and had no evidence for phase separation when modules with dissimilar endgroups were self-assembled to form "mixed monolayers". Our studies indicate that we can control the surface composition and density of displayed bioactive molecules by manipulating the bulk concentrations of the functionalized modules at the start of the self-assembly experiment. These molecularly engineered self-assembling protein surfaces provide a novel route for the display of large functionalized molecules, e.g. growth factors, at interfaces presenting notable innovation in SAM technology.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
