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Synthetic binder molecules that discriminate between isoforms of human Carbonic Anhydrase in blood.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
IFM Kemi, Linköpings Universitet.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
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(English)Manuscript (Other academic)
Abstract [en]

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.

URN: urn:nbn:se:uu:diva-101404OAI: oai:DiVA.org:uu-101404DiVA: diva2:212937
Available from: 2009-04-25 Created: 2009-04-25 Last updated: 2010-01-14
In thesis
1. Polypeptide Conjugates as High-affinity Binders for Proteins
Open this publication in new window or tab >>Polypeptide Conjugates as High-affinity Binders for Proteins
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A novel concept for protein recognition has been developed. The recognition unit is a hybrid molecule obtained by conjugation of a small organic molecule to a synthetic polypeptide selected from a 16-membered set of 42 amino acid residue sequences. The sequences are unordered and have no prior relation to the target proteins. The concept is based on the hypothesis that a small set of sequences capable of hydrophobic interactions, hydrogen bonding and electrostatic interactions can yield a binder for any selected protein, provided that the small molecule shows medium affinity or better and is reasonably selective.

The concept has been illustrated by the design, synthesis and evaluation of binders for three different proteins, the C-reactive protein, CRP, human Carbonic anhydrase II, HCAII, and Acetylcholine esterase, AChE. Highly efficient binders for CRP have been developed by conjugation of a derivative of the natural ligand, phosphocholine, to the side chain of one of the amino acids in each polypeptide. The binders in the set show a wide range of affinities for CRP and the tightest binder, 4-C10L17-PC6, binds almost irreversibly. Selected binders have been evaluated in human serum, where they capture CRP with high selectivity.High-affinity binders have been developed for HCAII, and the selectivity evaluated by extraction of the protein from blood. The binder 4-C37L34-B, a polypeptide conjugated to a spacered benzenesulphonamide residue, was able to extract Carbonic anhydrases specifically and to discriminate between the two isoforms of human Carbonic anhydrase. The conjugation of an acridine derivative to a polypeptide via a 14 atom spacer has been shown to yield a binder with high affinity and selectivity for AChE. The selectivity was demonstrated by extraction of AChE from Cerebrospinal fluid.

This thesis focuses on the development of a fast and reliable procedure for the construction, selection and evaluation of protein binders, with the ambition to develop a technology that is applicable to the development of binders for all proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 47 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 650
Protein recognition, polypeptide conjugates, peptide, molecular interactions, C-reactive protein, Acetylcholine esterase, Human Carboinc anhydrase
National Category
Chemical Sciences
Research subject
Organic Chemistry
urn:nbn:se:uu:diva-101406 (URN)978-91-554-7542-0 (ISBN)
Public defence
2009-06-12, B:21, BMC, Husargatan 3, Uppsala, 10:15 (English)
Available from: 2009-05-20 Created: 2009-04-25 Last updated: 2009-05-20Bibliographically approved

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