Direct immobilization of protein G variants with various numbers of cysteine residues on a gold surface
Direct immobilization of protein G variants with various numbers of cysteine residues on a gold surface
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DOI：
10.1021/ac0619231
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发表时间：
2007-04-01
影响因子：
7.4
通讯作者：
Chung, Bong Hyun
中科院分区：
文献类型：
--
作者：
Lee, Jeong Min;Park, Hyun Kyu;Chung, Bong Hyun
蛋白G是一种抗体结合蛋白，它专门针对抗体的Fc区。因此，在众多的免疫分析中，它被广泛用于固定不同类型的抗体。在这里，我们已经改造了链球菌蛋白G，使其在N端含有不同数量的半胱氨酸残基，从而在裸金表面形成定向良好的蛋白G膜。SPR和SPR成像分析表明，与未标记蛋白G相比，半胱氨酸标记蛋白G处理的金表面具有更好的抗体结合能力。AFM图像显示，抗体结合在半胱氨酸标记蛋白G表面的表面覆盖率高于完整蛋白G表面。半胱氨酸标记的蛋白G在金表面的正确定位也提供了更好的固定抗体的定位，从而增强了抗原检测。此外，在多轮抗体相互作用试验中，蛋白G表面保持了较高的抗体结合能力。本研究构建的半胱氨酸标记蛋白G可作为多种免疫传感器中定向抗体固定化的有价值的环节。
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.