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Title: Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies

Abstract

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39 amino-acid targeting sequence (Sil3T8) to direct a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundances in excess of 200,000 proteins per frustule. The fluorescence of either a derivative of trinitrotoluene (TNT) bound to the scFv or the endogenous fluorescence of EGFP was used to monitor pro-tein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. We find that proteins within isolated frustules undergo isotropic rotational motions with two-fold increases in rotational correlation times, which are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibod-ies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results argue that dramatic increases in protein conforma-tional stability within the biosilica frustulemore » matrices arise through molecular crowding, acting to retain native protein folds and associated functionality to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.« less

Authors:
 [1];  [2];  [2];  [3]; ORCiD logo [1]
  1. Department of Basic Medical Sciences, Western University of Health Sciences, Lebanon, Oregon 97355, United States
  2. Marine Biotechnology, Pacific Northwest National Laboratory, Sequim, Washington 98382, United States
  3. Marine Biotechnology, Pacific Northwest National Laboratory, Sequim, Washington 98382, United States; Department of Microbiology, Oregon State University, Corvallis, Oregon 97331, United States
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1422345
Report Number(s):
PNNL-SA-117286
Journal ID: ISSN 1043-1802; 400403909
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Bioconjugate Chemistry; Journal Volume: 28; Journal Issue: 11
Country of Publication:
United States
Language:
English
Subject:
Diatom; Biosilica

Citation Formats

Xiong, Yijia, Ford, Nicole R., Hecht, Karen A., Roesijadi, Guritno, and Squier, Thomas C.. Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies. United States: N. p., 2017. Web. doi:10.1021/acs.bioconjchem.7b00512.
Xiong, Yijia, Ford, Nicole R., Hecht, Karen A., Roesijadi, Guritno, & Squier, Thomas C.. Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies. United States. doi:10.1021/acs.bioconjchem.7b00512.
Xiong, Yijia, Ford, Nicole R., Hecht, Karen A., Roesijadi, Guritno, and Squier, Thomas C.. Thu . "Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies". United States. doi:10.1021/acs.bioconjchem.7b00512.
@article{osti_1422345,
title = {Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies},
author = {Xiong, Yijia and Ford, Nicole R. and Hecht, Karen A. and Roesijadi, Guritno and Squier, Thomas C.},
abstractNote = {Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39 amino-acid targeting sequence (Sil3T8) to direct a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundances in excess of 200,000 proteins per frustule. The fluorescence of either a derivative of trinitrotoluene (TNT) bound to the scFv or the endogenous fluorescence of EGFP was used to monitor pro-tein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. We find that proteins within isolated frustules undergo isotropic rotational motions with two-fold increases in rotational correlation times, which are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibod-ies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results argue that dramatic increases in protein conforma-tional stability within the biosilica frustule matrices arise through molecular crowding, acting to retain native protein folds and associated functionality to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.},
doi = {10.1021/acs.bioconjchem.7b00512},
journal = {Bioconjugate Chemistry},
number = 11,
volume = 28,
place = {United States},
year = {Thu Oct 12 00:00:00 EDT 2017},
month = {Thu Oct 12 00:00:00 EDT 2017}
}