title: Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter.
abstract: Human 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 VH 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.
authors.affiliation: Antibody Engineering, Mogam Biotechnology Research Institute, Yongin, Republic of Korea.
authors.forename: Dong-Sik
authors.initials: DS
authors.lastname: Kim
authors.affiliation: BioMedical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea.
authors.forename: Hyung-Nam
authors.initials: HN
authors.lastname: Song
authors.affiliation: Antibody Engineering, Mogam Biotechnology Research Institute, Yongin, Republic of Korea.
authors.forename: Hyo Jung
authors.initials: HJ
authors.lastname: Nam
authors.affiliation: Antibody Engineering, Mogam Biotechnology Research Institute, Yongin, Republic of Korea.
authors.forename: Sung-Geun
authors.initials: SG
authors.lastname: Kim
authors.affiliation: Antibody Engineering, Mogam Biotechnology Research Institute, Yongin, Republic of Korea.
authors.forename: Young-Seoub
authors.initials: YS
authors.lastname: Park
authors.affiliation: Antibody Engineering, Mogam Biotechnology Research Institute, Yongin, Republic of Korea.
authors.forename: Jae-Chan
authors.initials: JC
authors.lastname: Park
authors.affiliation: BioMedical Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
authors.affiliation: Antibody Engineering, Mogam Biotechnology Research Institute, Yongin, Republic of Korea.
authors.forename: Hyung-Kwon
authors.initials: HK
authors.lastname: Lim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D002942:Circular Dichroism; D022801:Complementarity Determining Regions / Q000737:chemistry; D019020:Directed Molecular Evolution / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005854:Germ Cells / Q000378:metabolism; D006801:Humans; D056724:Immunity, Humoral / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D012995:Solubility; D013312:Stress, Physiological; D013816:Thermodynamics
chemical_list: D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region