Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter

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dc.contributor.authorD S Kim-
dc.contributor.authorHyung Nam Song-
dc.contributor.authorH J Nam-
dc.contributor.authorS G Kim-
dc.contributor.authorY S Park-
dc.contributor.authorJ C Park-
dc.contributor.authorEui-Jeon Woo-
dc.contributor.authorH K Lim-
dc.date.accessioned2017-04-19T09:53:27Z-
dc.date.available2017-04-19T09:53:27Z-
dc.date.issued2014-
dc.identifier.issn1932-6203-
dc.identifier.uri10.1371/journal.pone.0098178ko
dc.identifier.urihttps://oak.kribb.re.kr/handle/201005/12005-
dc.description.abstractHuman immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one V H protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.-
dc.publisherPublic Library of Science-
dc.titleDirected evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter-
dc.title.alternativeDirected evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter-
dc.typeArticle-
dc.citation.titlePLoS One-
dc.citation.number6-
dc.citation.endPagee98178-
dc.citation.startPagee98178-
dc.citation.volume9-
dc.contributor.affiliatedAuthorHyung Nam Song-
dc.contributor.affiliatedAuthorEui-Jeon Woo-
dc.contributor.alternativeName김동식-
dc.contributor.alternativeName송형남-
dc.contributor.alternativeName남효정-
dc.contributor.alternativeName김성근-
dc.contributor.alternativeName박영섭-
dc.contributor.alternativeName박재찬-
dc.contributor.alternativeName우의전-
dc.contributor.alternativeName임형권-
dc.identifier.bibliographicCitationPLoS One, vol. 9, no. 6, pp. e98178-e98178-
dc.identifier.doi10.1371/journal.pone.0098178-
dc.description.journalClassY-
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Division of Biomedical Research > Disease Target Structure Research Center > 1. Journal Articles
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