title: Heat-induced irreversible denaturation of the camelid single domain VHH antibody is governed by chemical modifications.
abstract: The variable domain of camelid heavy chain antibody (VHH) is highly heat-resistant and is therefore ideal for many applications. Although understanding the process of heat-induced irreversible denaturation is essential to improve the efficacy of VHH, its inactivation mechanism remains unclear. Here, we showed that chemical modifications predominantly governed the irreversible denaturation of VHH at high temperatures. After heat treatment, the activity of VHH was dependent only on the incubation time at 90 °C and was insensitive to the number of heating (90 °C)-cooling (20 °C) cycles, indicating a negligible role for folding/unfolding intermediates on permanent denaturation. The residual activity was independent of concentration; therefore, VHH lost its activity in a unimolecular manner, not by aggregation. A VHH mutant lacking Asn, which is susceptible to chemical modifications, had significantly higher heat resistance than did the wild-type protein, indicating the importance of chemical modifications to VHH denaturation.
authors.affiliation: From the National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan and.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Akazawa-Ogawa
authors.affiliation: From the National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan and.
authors.forename: Mizuki
authors.initials: M
authors.lastname: Takashima
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Lee
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.affiliation: Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.affiliation: From the National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan and k-uegaki@aist.go.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Uegaki
authors.affiliation: From the National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan and hagihara-kappael@aist.go.jp.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Hagihara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001833:Body Temperature Regulation / Q000276:immunology*; D002162:Camelus; D006358:Hot Temperature; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D007140:Immunoglobulin Fab Fragments; D061905:Single-Domain Antibodies
keywords: Antibody; Antibody Engineering; Protein Aggregation; Protein Chemical Modification; Protein Denaturation; Protein Folding; Protein Stability
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