Synthesis and Characterization of Bioconjugates of S-Layer Proteins
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- 作者：Parthasarathy Sampathkumar and M. Lane Gilchrist ; Jr.
- 刊名：Bioconjugate Chemistry
- 出版年：2004
- 出版时间：July 2004
- 年：2004
- 卷：15
- 期：4
- 页码：685 - 693
- 全文大小：638K
- 年卷期：v.15,no.4(July 2004)
- ISSN：1520-4812
文摘
The self-assembling proteins that form crystalline surface layers (S-layers) on many microbial specieshave found numerous applications due to their nanostructured nature. To devise a new method toconstruct 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 infunction 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-tetheredendgroups on a surface. This paper examines the integration of protein purification, conjugation, andsurface assembly that has led to the development of this new method for the formation ofnanostructured surfaces. Purified S-layer proteins from Lactobacillus brevis were conjugated withsmall molecule probes and polymers using amine-based reactions. To keep multiple labeling of proteinamine groups to acceptable levels, the conjugations were performed at pH 6.5, allowing for limitedyields (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 protocolfor 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 homogeneousdistributions of tethered molecules. Also, in cases where the modular assembly of two distinct typesof 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 asthe starting assembly conditions guide displayed endgroup concentrations in mixed molecularmonolayers.