Synthetic binder molecules that discriminate between isoforms of human Carbonic Anhydrase in blood.
(English)Manuscript (Other academic)
From a 16-membered set of 42 residue polypeptide conjugates designed to bind human Carbonic Anhydrases (HCA), two binder candidates 4-C37L34-B och 3-C15L8-B were shown to bind the isoform HCAII with high affinity in a fluorescence based screening assay. To determine their specificity for Carbonic Anhydrases the binders were immobilized on polystyrene beads and submerged in lysed blood, washed three times, cleaved from the beads, analyzed by SDS-PAGE and shown to specifically extract Carbonic Anhydrases from the complex biological mixture. Two Carbonic Anhydrase isoforms with 60% homology exist in human blood with HCAI being present in 5-7 fold excess over HCAII and the ability of the binder molecules to discriminate between HCAI and HCAII was investigated. Due to the high degree of homology HCAI and HCAII could not be separated by electrophoresis and the instead affinities were determined by SPR biosensor analysis. The polypeptide conjugate 4-C37L34-B bound HCAII with a KD of 12 nM whereas it was 90 nM for the binding of HCAI, a ratio of 7.5. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two Carbonic Anhydrases were X and Y. This demonstration of selectivity between two very similar proteins is conspicuous in view of the fact that the molecular weight of each one of the binder molecules is little more than 5000, the fold is unordered and the polypeptide sequences are designed from scratch and have no prior relationship to Carbonic Anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.
IdentifiersURN: urn:nbn:se:uu:diva-101404OAI: oai:DiVA.org:uu-101404DiVA: diva2:212937