Skip to main content
. Author manuscript; available in PMC: 2010 May 8.
Published in final edited form as: J Mol Biol. 2009 Mar 13;388(3):443–461. doi: 10.1016/j.jmb.2009.03.016

Fig. 8.

Fig. 8

Generation of bispecific 3D5×C6.5 scDb adapter. (a) The recombinant 3D5×C6.5 gene was assembled to encode the following elements: leader signal from murine Ig kappa-chain (Leader), HA epitope (H), variable light chain of 3D5 scFv (VL3D5), 5-aa linker (short linker 1), variable heavy chain of C6.5 scFv (VHC6.5), 15-aa (G4S)3 linker (long linker), variable light chain of C6.5 scFv (VLC6.5), 5-aa linker (short linker 2), variable heavy chain of 3D5 scFv (VH3D5), and Strep-tag II peptide (S). The constructed gene was placed under the transcriptional control of the CMV promoter to produce the scDb in mammalian cells. (b) Schematic representation of 3D5×C6.5 polypeptide folded head-to-tail into a diabody-like structure to form antigen–binding sites for the C-terminal oligo-His tag and the c-erbB2 oncoprotein. (c) The secreted 3D5×C6.5 scDb was purified on Strep-Tactin affinity column and the protein characterized by western blot using an anti-HA mAb (lanes 2 and 3) and Strep-Tactin (lanes 5 and 6), both conjugated with AP. The samples in lanes 2 and 5 were boiled to denature the scDb protein to monomers, while proteins in lanes 3 and 6 were not denatured. The numbers on the left indicate molecular masses of protein standards (lanes 1 and 4) in kDa.