Fig. 2. Expression of the 372 and 373 scFvs in E. coli. (A) Western blot analysis of the purified His6-tagged scFv 372 (lane 1) and 373 (lane 2) obtained by affinity chromatography. The arrow points to the scFv protein. The migration of molecular weight markers (in kDa) is shown on the left. (B) An ELISA to measure and compare the binding activity of purified scFv fragments and the parental Ig. A solution of 10 µg/ml of HP1ß-GST protein (dark columns) and 3 mg/ml of lysozyme (light columns) was used for coating. As a negative control, a purified anti-lysozyme D1.3 scFv fragment was used. Values obtained by coating the wells with GST alone have been subtracted. (C) An ELISA to determine the fine specificity of the scFv fragments against recombinant chromodomain-containing proteins HP1ß, HP1, HP1, mPc1 and the chromodomain (CD) peptide, all fused to GST. Values obtained by coating the wells with GST alone have been subtracted. (D) An ELISA to test competition of anti-chromodomain scFv fragments against the same epitope. Miniwells were coated with soluble purified 372 (upper panel) or the 373 scFv fragment (lower panel). HP1ß-GST protein, which had been previously incubated for 30 minutes at room temperature with or without the purified anti-CD scFv fragments or the non relevant anti-lysozyme D1.3 scFv at a 1:10 molar ratio, was added to the coated wells. The HP1ß-GST bound protein was determined by further incubation with anti-GST antibodies and anti-goat-peroxidase.

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