Direct immobilization of protein g variants with various numbers of cysteine residues on a gold surface.

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.