title: Direct immobilization of protein g variants with various numbers of cysteine residues on a gold surface.
abstract: Protein G is an antibody binding protein, which specifically targets the Fc region of an antibody. It therefore has been widely used to immobilize different types of antibodies in numerous immunoassays. Here, we have engineered Streptococcus protein G to contain various numbers of cysteine residues at the N-terminus and therefore to form well-oriented protein G films on bare gold. SPR and SPR imaging analyses indicated that a gold surface treated with cysteine-tagged protein G possesses a superior antibody binding ability compared to one treated with tag-free protein G. AFM images indicated a higher surface coverage by antibody binding on the cysteine-tagged protein G surface than the intact protein G surface. The proper orientation of cysteine-tagged protein G on a gold surface also afforded better orientation of immobilized antibodies, resulting in enhanced antigen detection. Moreover, the protein G surfaces maintained their high antibody binding ability during multiple rounds of antibody interaction tests. The cysteine-tagged protein G constructed in this study can be a valuable link for oriented antibody immobilization in a variety of immunosensors.
authors.affiliation: BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yuseong, Daejeon 305-600, Korea.
authors.forename: Jeong Min
authors.initials: JM
authors.lastname: Lee
authors.forename: Hyun Kyu
authors.initials: HK
authors.lastname: Park
authors.forename: Yongwon
authors.initials: Y
authors.lastname: Jung
authors.forename: Jin Kyeong
authors.initials: JK
authors.lastname: Kim
authors.forename: Sun Ok
authors.initials: SO
authors.lastname: Jung
authors.forename: Bong Hyun
authors.initials: BH
authors.lastname: Chung
mesh_terms: D000327:Adsorption; D000906:Antibodies / Q000276:immunology; D000937:Antigen-Antibody Reactions; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D018625:Microscopy, Atomic Force / Q000379:methods; D053758:Nanoparticles / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013291:Streptococcus / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods; D013499:Surface Properties
chemical_list: D000906:Antibodies; C029152:G-substrate; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D006046:Gold; D003545:Cysteine