Analysis of direct immobilized recombinant protein G on a gold surface.

For the immobilization of IgG, various techniques such as chemical linker, thiolated protein G methods, and fragmentation of antibodies have been reported [Y.M. Bae, B.K. Oh, W. Lee, W.H. Lee, J.W. Choi, Biosensors Bioelectron. 21 (2005) 103; W. Lee, B.K. Oh, W.H. Lee, J.W. Choi, Colloids Surf. B-Biointerfaces, 40 (2005) 143; A.A. Karyakin, G.V. Presnova, M.Y. Rubtsova, A.M. Egorov, Anal. Chem. 72 (2000) 3805]. Here, we modified the immunoglobulin Fc-binding B-domain of protein G to contain two cysteine residues at its C-terminus by a genetic engineering technique. The resulting recombinant protein, RPGcys, retained IgG-binding activity in the same manner as native protein G. RPGcys was immobilized on a gold surface by strong affinity between thiol of cysteine and gold. The orientations of both IgG layers immobilized on the base recombinant protein Gs were analyzed by fluorescence microscope, atomic force microscope (AFM), and surface plasmon resonance (SPR). Our data revealed that IgG-binding activity of RPGcys on gold surface significantly increased in comparison to wild type of protein G (RPGwild), which was physically adsorbed due to absence of cysteine residue. Immobilization of highly oriented antibodies based on cysteine-modified protein G could be useful for the fabrication of immunosensor systems.

[1]  Shaoyi Jiang,et al.  Controlling Antibody Orientation on Charged Self-Assembled Monolayers , 2003 .

[2]  Nikin Patel,et al.  Immobilization of Protein Molecules onto Homogeneous and Mixed Carboxylate-Terminated Self-Assembled Monolayers , 1997 .

[3]  M Aizawa,et al.  Assembling of engineered IgG-binding protein on gold surface for highly oriented antibody immobilization. , 2000, Journal of biotechnology.

[4]  B. Chin,et al.  Rapid and sensitive biosensor for Salmonella. , 2000, Biosensors & bioelectronics.

[5]  G J Kleywegt,et al.  Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. , 1995, Structure.

[6]  R. Collins,et al.  Correlation of real time spectroellipsometry and atomic force microscopy measurements of surface roughness on amorphous semiconductor thin films , 1996 .

[7]  Jeong-Woo Choi,et al.  Detection of Escherichia coli O157:H7 using immunosensor based on surface plasmon resonance , 2002 .

[8]  Characterization of IgG Langmuir-Blodgett films immobilized on functionalized polymers. , 2000, Talanta.

[9]  Paul W. Bohn,et al.  Cysteine-specific surface tethering of genetically engineered cytochromes for fabrication of metalloprotein nanostructures , 1994 .

[10]  M. Meyerhoff,et al.  Separation-free sandwich enzyme immunoassays using microporous gold electrodes and self-assembled monolayer/immobilized capture antibodies. , 1994, Analytical chemistry.

[11]  M. Smyth,et al.  Oriented immobilization of antibodies and its applications in immunoassays and immunosensors. , 1996, The Analyst.

[12]  Fumio Mizutani,et al.  Site-specific immobilization of molecularly engineered dihydrofolate reductase to gold surfaces , 1994 .

[13]  Martin Hegner,et al.  Ultralarge atomically flat template-stripped Au surfaces for scanning probe microscopy , 1993 .

[14]  D. O'Shannessy,et al.  Site-specific immobilization of antibodies by their oligosaccharide moieties to new hydrazide derivatized solid supports. , 1988, Journal of immunological methods.

[15]  Vadim L. Chashchin,et al.  A model of optical immunosensor for hemoproteins based on Langmuir-Blodgett films of FITC-labelled immunoglobulin G , 2000 .

[16]  M C Davies,et al.  Surface plasmon resonance for real time in situ analysis of protein adsorption to polymer surfaces. , 1997, Biomaterials.

[17]  S. Fahnestock.  Cloned streptococcal protein G genes , 1987 .