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35302520151050-5-10100 200 300 400 500 600 700 800 900 1000 1100TimeTable 1. SPR kinetic variables calculated using 1:1 Langmuir binding modelVariable Value Unitk a 1.1 x 10 6 1/Msk d 2.1 x 10 -3 1/sK D , synthetic 1.9 x 10 -9 MK D , recombinant 0.5 x 10 -9 MAnistropy (x1000)1751701651601550 50 100 150 200 250[human PDGFR beta] (nM)Fig. 2. FP plot of labelled synthetic Affibody.CD[mdeg]90500-50-60180 200 220 240260Wavelength [nm]Fig. 3. CD spectra of synthetic (solid line) andrecombinant (broken line) Affibody.Resp. Diff.Fig. 4. SPR sensorgrams for syntheticAffibody.Circular dichroism (CD) spectra wererecorded at ambient temperature on a JascoJ-715 spectropolarimeter equipped with a0.1 cm 100-QS Supracil cuvette. Theconcentration of Affibody was 0.2 mg/mLphosphate buffer (pH 7.4). Synthetic andrecombinant Affibody gave similarspectra, both showing characteristic alphahelixfeatures (Figure 3).Surface plasmon resonance (SPR)analyses was performed on a BiaCore3000 equipped with a CM5 chip activatedusing EDC and NHS. The rhPDGFR βprotein (10 µg/mL acetate buffer, pH 4.0)was passed over the surface for 7 min atflow rate 10 µL/min using 10 mM HEPESbuffer containing NaCl, EDTA andsurfactant P20 (pH 7.4). Affinity andkinetic study were performed atimmobilisation of 1.6 µg/mL rhPDGFR βat the response level 3100 RU. Theconcentration range of Affibody was 0.01–1 µg/mL. Each concentration was injectedin triplicate. Injection time was 3 min at aflow rate of 30 µL/min, dissociation timewas 15 min (Figure 4). The K D value forthe synthetic Affibody obtained from theSPR kinetics was similar to the K D valuereported for the recombinant counterpart(Table 1).ConclusionThe PDGFR specific Affibody wassynthesised in good purity and acceptableyield. The synthetic Affibody wascomparable with its recombinantcounterpart with respect to secondarystructure content and biological activity.References1. Marino, M.E., et al. U.S. Patent Appl. 20090191124 (2009).2. Palasek, S.A., et al. J. Pept. Sci. 13, 143-148 (2007).3. Samples of recombinant PDGFR specific Affibody and K D data for recombinant PDGFR specificAffibody were provided by Affibody AB, Sweden.4. Nord, K., et al. Nature Biotechnol. 15, 772-777 (1997).5. Arora, P., et al. Protein Sci. 13, 847-853 (2004).63