Redesigning of anti-c-Met single chain Fv antibody for the cytoplasmic folding and its structural analysis.
Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition.
Edwardraja
Selvakumar
S
Department of Chemical and Biochemical Engineering, Pusan National University, Busan, South Korea.
Neelamegam
Rameshkumar
R
Ramadoss
Vijayaraj
V
Venkatesan
Subramanian
S
Lee
Sun-Gu
SG
D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D019859:Proto-Oncogene Proteins c-met / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism
D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D019859:Proto-Oncogene Proteins c-met