skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports

Abstract

Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.

Authors:
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
940515
Report Number(s):
UCRL-JRNL-228513
Journal ID: ISSN 0006-3525; BIPMAA; TRN: US200824%%78
DOE Contract Number:
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biopolymers, vol. 90, no. 3, January 1, 2008, pp. 450-458; Journal Volume: 90; Journal Issue: 3
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADSORPTION; CROSS-LINKING; PROTEINS; SPACERS

Citation Formats

Camarero, J A. Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports. United States: N. p., 2007. Web.
Camarero, J A. Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports. United States.
Camarero, J A. Wed . "Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports". United States. doi:. https://www.osti.gov/servlets/purl/940515.
@article{osti_940515,
title = {Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports},
author = {Camarero, J A},
abstractNote = {Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.},
doi = {},
journal = {Biopolymers, vol. 90, no. 3, January 1, 2008, pp. 450-458},
number = 3,
volume = 90,
place = {United States},
year = {Wed Feb 21 00:00:00 EST 2007},
month = {Wed Feb 21 00:00:00 EST 2007}
}
  • Protein microarrays have emerged as important tools for screening protein-protein interactions and hold great potential for various applications including proteomics research, drug discovery, and diagnostics. This work describes a novel method for the traceless immobilization of proteins to a solid support through split-intein mediated protein trans-splicing. This method has been successfully used for the immobilization of biologically active proteins from very diluted samples ({approx}1{micro}M) and it does not require the purification of the protein to be attached. This makes possible the direct immobilization of proteins from complex mixtures such as cellular lysates and it can also be easily interfaced withmore » cell-free expression systems for high-throughput production of protein microarrays.« less
  • The site-specific immobilization of histidine-tagged proteins to patterns formed by far-field and near-field exposure of films of aminosilanes with protein-resistant photolabile protecting groups is demonstrated. After deprotection of the aminosilane, either through a mask or using a scanning near-field optical microscope, the amine terminal groups are derivatized first with glutaraldehyde and then with N-(5-amino-1-carboxypentyl)iminodiacetic acid to yield a nitrilo-triacetic-acid-terminated surface. After complexation with Ni 2+, this surface binds histidine-tagged GFP and CpcA-PEB in a site-specific fashion. The chemistry is simple and reliable and leads to extensive surface functionalization. Bright fluorescence is observed in fluorescence microscopy images of micrometer- and nanometer-scalemore » patterns. X-ray photoelectron spectroscopy is used to study quantitatively the efficiency of photodeprotection and the reactivity of the modified surfaces. The efficiency of the protein binding process is investigated quantitatively by ellipsometry and by fluorescence microscopy. We find that regions of the surface not exposed to UV light bind negligible amounts of His-tagged proteins, indicating that the oligo(ethylene glycol) adduct on the nitrophenyl protecting group confers excellent protein resistance; in contrast, exposed regions bind His-GFP very effectively, yielding strong fluorescence that is almost completely removed on treatment of the surface with imidazole, confirming a degree of site-specific binding in excess of 90%. As a result, this simple strategy offers a versatile generic route to the spatially selective site-specific immobilization of proteins at surfaces.« less
  • The site-specific immobilization of histidine-tagged proteins to patterns formed by far-field and near-field exposure of films of aminosilanes with protein-resistant photolabile protecting groups is demonstrated. After deprotection of the aminosilane, either through a mask or using a scanning near-field optical microscope, the amine terminal groups are derivatized first with glutaraldehyde and then with N-(5-amino-1-carboxypentyl)iminodiacetic acid to yield a nitrilo-triacetic-acid-terminated surface. After complexation with Ni 2+, this surface binds histidine-tagged GFP and CpcA-PEB in a site-specific fashion. The chemistry is simple and reliable and leads to extensive surface functionalization. Bright fluorescence is observed in fluorescence microscopy images of micrometer- and nanometer-scalemore » patterns. X-ray photoelectron spectroscopy is used to study quantitatively the efficiency of photodeprotection and the reactivity of the modified surfaces. The efficiency of the protein binding process is investigated quantitatively by ellipsometry and by fluorescence microscopy. We find that regions of the surface not exposed to UV light bind negligible amounts of His-tagged proteins, indicating that the oligo(ethylene glycol) adduct on the nitrophenyl protecting group confers excellent protein resistance; in contrast, exposed regions bind His-GFP very effectively, yielding strong fluorescence that is almost completely removed on treatment of the surface with imidazole, confirming a degree of site-specific binding in excess of 90%. As a result, this simple strategy offers a versatile generic route to the spatially selective site-specific immobilization of proteins at surfaces.« less