Disulfide linkage engineering for improving biophysical properties of human VH domains

From National Research Council Canada

DOI	Resolve DOI: https://doi.org/10.1093/protein/gzs055
Author	Search for: Kim, D.Y.¹; Search for: Kandalaft, H.¹; Search for: Ding, W.¹; Search for: Ryan, S.¹; Search for: Van Faassen, H.¹; Search for: Hirama, T.¹; Search for: Foote, S.J.¹; Search for: MacKenzie, R.¹; Search for: Tanha, J.¹
Affiliation	National Research Council of Canada. Human Health Therapeutics
Format	Text, Article
Subject	Affinity reagents; Biophysical properties; Conformational change; Disulfide linkages; Heavy chain; Heavy-chain antibodies; Human antibodies; Model domains; Multi-angle light scatterings; Therapeutic potentials; thermostability; Variable domain; Agglomeration; Amino acids; Antibodies; Libraries; Turbidity; Sulfur compounds; antibody; cysteine; disulfide; heavy chain antibody variable domain; protein A; unclassified drug; amino acid substitution; Artiodactyla; binding affinity; biophysics; circular dichroism; covalent bond; disulfide linkage; gel permeation chromatography; mass spectrometry; molecular cloning; protein engineering; protein expression; thermostability; turbidity; Amino Acid Sequence; Amino Acid Substitution; Disulfides; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Molecular Sequence Data; Protein Conformation; Protein Engineering; Protein Stability; Sequence Alignment; Temperature; Camelidae
Abstract	To enhance their therapeutic potential, human antibody heavy chain variable domains (VHs) would benefit from increased thermostability. The highly conserved disulfide linkage that connects Cys23 and Cys104 residues in the core of VH domains is crucial to their stability and function. It has previously been shown that the introduction of a second disulfide linkage can increase the thermostability of camelid heavy-chain antibody variable domains (VHHs). Using four model domains we demonstrate that this strategy is also applicable to human VH domains. The introduced disulfide linkage, formed between Cys54 and Cys78 residues, increased the thermostability of VHs by 1418°C. In addition, using a novel hexa-histidine capture technology, circular dichroism, turbidity, size exclusion chromatography and multiangle light scattering measurements, we demonstrate reduced VH aggregation in domains with the Cys54Cys78 disulfide linkage. However, we also found that the engineered disulfide linkage caused conformational changes, as indicated by reduced binding of the VHs to protein A. This indicates that it may be prudent to use the synthetic VH libraries harboring the engineered disulfide linkage before screening for affinity reagents. Such strategies may increase the number of thermostable binders. © 2012 The Author.
Publication date	2012
In	Protein Engineering, Design and Selection 25, no. 10 (2012): 581–589.
Language	English
Peer reviewed	Yes
NPARC number	21269166
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Record identifier	058a7ad7-aa5d-46f3-bb92-5b6891a2747d
Record created	2013-12-12
Record modified	2020-04-21

Date modified:: 2024-04-18