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.
Department of Chemical and Biomolecular Engineering, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea.
Hyunhee
H
Kim
Da-Yeon
DY
Kang
Hyun-Jeong
HJ
Goh
Byung-Keun
BK
Oh
Ravindra P
RP
Singh
Soo-Min
SM
Oh
Jeong-Woo
JW
Choi
D000327:Adsorption; D005818:Genetic Engineering / Q000379:methods*; D006046:Gold / Q000737:chemistry*; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D018625:Microscopy, Atomic Force / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013499:Surface Properties
C029152:G-substrate; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D006046:Gold