Analysis of direct immobilized recombinant protein G on a gold surface |
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Authors: | Kim Hyunhee Kang Da-Yeon Goh Hyun-Jeong Oh Byung-Keun Singh Ravindra P Oh Soo-Min Choi Jeong-Woo |
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Affiliation: | aDepartment of Chemical and Biomolecular Engineering, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea;bInterdisciplinary Program of Integrated Biotechnology, Sogang University, Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea;cResearch Institute for Applied Science and Technology, Sogang University , Shinsu-dong, Mapo-gu, Seoul 121-742, Republic of Korea |
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Abstract: | 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. |
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Keywords: | Protein G Genetic engineering AFM Gold surface |
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