Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A
Article first published online: 20 DEC 2001
European Journal of Biochemistry
Volume 268, Issue 15, pages 4269–4277, August 2001
How to Cite
Nord, K., Nord, O., Uhlén, M., Kelley, B., Ljungqvist, C. and Nygren, P.-Å. (2001), Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A. European Journal of Biochemistry, 268: 4269–4277. doi: 10.1046/j.1432-1327.2001.02344.x
- Issue published online: 20 DEC 2001
- Article first published online: 20 DEC 2001
- (Received 9 February 2001, revised 31 May 2001, accepted 7 June 2001)
- affibody ligand;
- affinity maturation;
- affinity chromatography;
- coagulation factor VIII;
- combinatorial phage library.
Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an α helical bacterial receptor domain derived from staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used during competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation constants (Kd) in the range 100–200 nm as determined by biosensor analyses. One variant (ZrVIII:3, 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and selective recovery of biologically active rVIII from Chinese hamster ovary cell supernatant-derived feed stocks. The purity of the enriched rVIII was comparable with rVIII material purified by immunoaffinity chromatography using a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapping showed that the monoclonal antibody and the affibody ligand competed for the same or at least overlapping epitopes on rVIII. In addition, the ZrVIII:3 variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content similar to that of the parental Z domain used as scaffold. In biosensor studies, the synthetic affibody was immobilized recruiting the C-terminal cysteine residue, and demonstrated to bind both recombinantly produced and plasma-derived factor VIII. From a secondary library, constructed by re-randomization of relevant positions identified after alignment of the first-generation variants, a panel of affinity-improved second-generation affibodies were selected of which one clone showed a dissociation constant (Kd) for rVIII of 5 nm. Several of these variants also showed higher apparent binding efficiencies towards rVIII when analyzed as immobilized ligands in biosensor experiments. Taken together, the results suggest that affibody ligands produced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or monitoring tools.