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Title 

Covalent and oriented surface immobilization of antibody using photoactivatable antibody Fc-binding protein expressed in Escherichia coli

 

대장균 생산 광활성 항체 Fc 결합 단백질을 이용한 고배향 항체 공유 결합 고정

Authors 

Yeolin LeeJiyun JeongGabi LeeJeong Hee MoonMyung Kyu Lee

Publisher 

American Chemical Society

Issue Date 

2016

Citation 

Analytical Chemistry, vol. 88, no. 19, pp. 9503-9509

Abstract 

Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.

ISSN 

0003-2700

Link 

http://dx.doi.org/10.1021/acs.analchem.6b02071

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2019-05-02


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