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Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli
journal contribution
posted on 2016-08-30, 00:00 authored by Yeolin Lee, Jiyun Jeong, Gabi Lee, Jeong
Hee Moon, Myung Kyu LeeFc-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.
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Escherichia coli Fc-specific antibody binding proteinsUVsupport protein expressionMRS 5m Coexpressionphotoactivatable FcBP overexpressionPhotoactivatable Antibody Fc-Binding ProteinOriented Surface Immobilizationreport novel photoactivatable FcBPsmethionyl tRNA synthetasephoto-cross-linked antibody arraysseraN-terminal cysteine residuesMRS 5m proteinsphotoactivatable FcBPs6 xHis tag
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