VH/VL interface engineering to promote selective expression and inhibit conformational isomerization of thrombopoietin receptor agonist single-chain diabody

Protein Eng Des Sel. 2010 Aug;23(8):667-77. doi: 10.1093/protein/gzq034. Epub 2010 Jun 24.


Thrombopoietin receptor agonist humanized VB22B single-chain diabody (hVB22B (scFv)(2)) was found to be expressed as a mixture of two conformational isomers, a single-chain diabody form and a bivalent scFv form, which had different V(H)/V(L) (variable region of the heavy chain/light chain) association patterns. The single-chain diabody form showed significantly higher biological activity than the bivalent scFv form and, when incubated at elevated temperatures, exhibited novel isomerization to the inactive bivalent scFv form. Therefore, therapeutic development of hVB22B (scFv)(2) would require separation of the purified single-chain diabody form from the mixture of the two conformational isomers and also inhibition of isomerization into an inactive bivalent scFv form during storage. Novel V(H)/V(L) interface engineering in hVB22 (scFv)(2), in which hydrogen bonding between H39 and L38 was substituted with electrostatic interaction to enhance the desired V(H)/V(L) association and inhibit the undesired V(H)/V(L) association, enabled selective expression of the desired conformational isomer without any reduction in biological activity or thermal stability. Moreover, V(H)/V(L) interface-engineered hVB22 (scFv)(2) was completely resistant to isomerization. Because the hydrogen bonding interaction between H39 and L38 and the surrounding residues are highly conserved in human antibody sequences, V(H)/V(L) interface engineering could be generally applied to various (scFv)(2) molecules for selective expression and inhibition of the isomerization of conformational isomers.

MeSH terms

  • Animals
  • CHO Cells
  • Chromatography, Gel
  • Cricetinae
  • Cricetulus
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Hydrogen-Ion Concentration
  • Isomerism
  • Protein Conformation
  • Protein Engineering / methods*
  • Protein Stability
  • Receptors, Thrombopoietin / agonists
  • Receptors, Thrombopoietin / chemistry
  • Receptors, Thrombopoietin / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism*
  • Single-Chain Antibodies / chemistry
  • Single-Chain Antibodies / genetics
  • Single-Chain Antibodies / isolation & purification
  • Single-Chain Antibodies / metabolism*
  • Sodium Chloride
  • Surface Properties
  • Temperature


  • Receptors, Thrombopoietin
  • Recombinant Proteins
  • Single-Chain Antibodies
  • MPL protein, human
  • Sodium Chloride