Epitope mapping of a monoclonal antibody directed against the α-subunit of phosphofructokinase-1 from Saccharomyces cerevisiae by screening phage display libraries
✍ Scribed by Margrit Hollborn; Jürgen Kirchberger; Gerd Birkenmeier; Gerhard Kopperschläger
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 90 KB
- Volume
- 12
- Category
- Article
- ISSN
- 0952-3499
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✦ Synopsis
Phosphofructokinase-1 from Saccharomyces cerevisiae is composed of two types of subunits, a and b. Subunit-specific monoclonal antibodies were raised to elucidate structural and functional properties of both subunits. One monoclonal antibody, a-F3, binds to an epitope either at the C-terminal or at the N-terminal part of the a-polypeptide chain. By screening a heptapeptide library with this monoclonal antibody, a set of heptapeptides was selected, which contained the consensus sequences D-A-F and D-S-F. Two heptapeptides with these motifs were synthesized in order assess their capacity to inhibit the binding of antibody a-F3 to native phosphofructokinase-1. The peptide G-I-K-D-A-F-L inhibited the binding more strongly (IC 50 = 1.5 M) than (IC 50 = 33.3 M). Sequence matching revealed the presence of the D-A-F motif in the polypeptide chain of phosphofructokinase-1 at amino acid position 172-174. As a control, the nonapeptide A-P-T-S-K-D-A-F-L which corresponds to the sequence of the putative epitope was tested in the inhibition assay. In view of the high inhibitory capacity (IC 50 = 0.3 M) it was concluded that this nonapeptide represents the continuous epitope of phosphofructokinase-1 that is recognized by antibody a-F3.