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Evaluation of a noncanonical Cys40‐Cys55 disulfide linkage for stabilization of single‐domain antibodies
Author(s) -
Kim Dae Young,
Kandalaft Hiba,
Hussack Greg,
Raphael Shalini,
Ding Wen,
Kelly John F.,
Henry Kevin A.,
Tanha Jamshid
Publication year - 2019
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3595
Subject(s) - thermostability , disulfide linkage , chemistry , single domain antibody , disulfide bond , circular dichroism , crystallography , solubility , denaturation (fissile materials) , stereochemistry , antibody , biochemistry , cysteine , biology , nuclear chemistry , enzyme , genetics
Incorporation of noncanonical disulfide linkages into single‐domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures ( T m s), reversibility of thermal unfolding, solubility, and antigen‐binding affinities of three types of sdAbs (V H H, V H , and V L domains). The Cys40‐Cys55 disulfide linkage was tolerated by 9/9 V H Hs, 12/12 V H s, and 2/11 V L s tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40‐Cys55 disulfide linkage increased sdAb T m by an average of 10.0°C (range: 0–21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen‐binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.