An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly

FEBS Letters

Wörn, Arne¹  Plückthun, Andreas¹ 

[1]Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland

关键词: Disulfide bond;    ScFv fragment;    Intrabody;    Antibody engineering;    Protein stability;    AP;    alkaline phosphatase;    CDR;    complementarity determining region;    DTT;    dithiothreitol;    EDTA;    ethylenediaminetetraacetic acid;    ELISA;    enzyme linked immunosorbent assay;    FU;    fraction unfolded;    GdnHCl;    guanidinium hydrochloride;    hu;    humanized;    IMAC;    immobilized metal ion affinity chromatography;    IPTG;    isopropyl-β-d-thiogalactopyranoside;    K D;    equilibrium dissociation constant;    PBS;    phosphate-buffered saline;    PBST;    PBS with 0.05% Tween-20;    PCR;    polymerase chain reaction;    p185HER2-ECD;    extracellular domain of human epidermal growth factor receptor-2;    scFv;    antibody single-chain fragment;    SDS;    sodium dodecyl sulfate;    TBS;    Tris-buffered saline;    TBST;    TBS with 0.05% Tween-20;    V H;    variable domain of the heavy chain;    V L;    variable domain of the light chain;    4D5-SS+;    oxidized form of 4D5 scFv with cysteines;    4D5-SSred;    reduced dithiol form of 4D5 scFv with cysteines;    4D5-SS−;    cysteine-free variant of 4D5 scFv;   

DOI  :  10.1016/S0014-5793(98)00463-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A fully functional cysteine-free derivative of the intrinsically stable anti-HER2 scFv fragment hu4D5–8 was generated by replacing the disulfide forming cysteine residues in V _H and V _L with the amino acid combination valine-alanine in both domains. The antigen binding properties, determined by ELISA and BIAcore measurements, were not affected by removal of the disulfide bonds. The thermodynamic stability of the disulfide-containing scFv of 8.1 kcal/mol is decreased upon complete reduction of both disulfides to 2.7 kcal/mol, while that of the valine-alanine variant is somewhat higher (about 3.8 kcal/mol). Our results suggest that, in principle, a disulfide-free fully functional derivative of any scFv can be obtained, as long as the corresponding disulfide-containing scFv has a high enough thermodynamic stability.

【 授权许可】

Unknown