10.1021/acs.analchem.6b02071.s001
Yeolin Lee
Yeolin
Lee
Jiyun Jeong
Jiyun
Jeong
Gabi Lee
Gabi
Lee
Jeong
Hee Moon
Jeong
Hee
Moon
Myung Kyu Lee
Myung Kyu
Lee
Covalent and Oriented Surface Immobilization of Antibody
Using Photoactivatable Antibody Fc-Binding Protein Expressed in <i>Escherichia coli</i>
American Chemical Society
2016
Escherichia coli Fc-specific antibody binding proteins
UV
support protein expression
MRS 5m Coexpression
photoactivatable FcBP overexpression
Photoactivatable Antibody Fc-Binding Protein
Oriented Surface Immobilization
report novel photoactivatable FcBPs
methionyl tRNA synthetase
photo-cross-linked antibody arrays
sera
N-terminal cysteine residues
MRS 5m proteins
photoactivatable FcBPs
6 xHis tag
2016-08-30 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Covalent_and_Oriented_Surface_Immobilization_of_Antibody_Using_Photoactivatable_Antibody_Fc-Binding_Protein_Expressed_in_i_Escherichia_coli_i_/3827514
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 <i>E. coli</i>, which induce Fc-specific photo-cross-linking
with antibodies upon UV irradiation. Unfortunately, pMet did not support
protein expression in the native <i>E. coli</i> system,
and therefore we also developed an engineered methionyl tRNA synthetase
(MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable
FcBP overexpression in methionine-auxotroph <i>E. coli</i> 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.