| 1. |
- T. Tegler, Lotta, et al.
(författare)
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A highly selective polypeptide binder for human Acetylcholine esterase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A highly selective high-affinity polypeptide conjugate binder for human Acetylcholine Esterase (hAChE) has been obtained by coupling a derivative of acridine, a known medium-affinity inhibitor of hAChE, to each member of a 16-membered set of 42-residue polypeptide scaffolds. The best candidate, 4-C10L17-Ac, was identified to have a KD of 10 nM or less in an assay where each polypeptide conjugate was titrated with hAChE in 50 mM phosphate buffer at pH 7.0 and 298K. It was found in a sandwich ELISA to have high selectivity for hAChE in cerebrospinal fluid. Targeting the active site of hAChE by a polypeptide conjugate binder presents a special problem as it is buried deep inside the protein in a cavity that is approximately 20 Å deep. In order to permit simultaneous and cooperative binding of the acridine and the polypeptide to the active site and the AChE surface a fourteen atom spacer was needed. The 9-aminoacridine group was linked to the side chain of a lysine residue in each polypeptide via a spacer prepared from two aminohexanoic acid fragments. The results reinforce the impression that polypeptide conjugates are excellent alternatives to currently used protein binder technologies in diagnostic and therapeutic applications and that the conjugation of enzyme inhibitors to polypeptide scaffolds is a strategy of general applicability in the design of high-affinity binders for enzymes.
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| 2. |
- T. Tegler, Lotta, et al.
(författare)
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Efficient protein binders for the C-reactive protein from a designed chemically modified peptide library
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A polypeptide conjugate synthesized by coupling a small organic molecule to the side chain of an amino acid residue in a designed 42-residue polypeptide binds the C-reactive protein (CRP) essentially irreversibly. The specificity in human serum is equal to that of an avian antibody although the size is only 1/30 and the structure unordered. The polypeptide conjugate binds CRP several orders of magnitude more tightly than the small molecule due to the fact that one amino acid has been modified to include a more strongly interacting side chain. The polypeptide was selected from a 16-membered set of sequences with no prior relationship to the target protein and designed to fold into a helix-loop-helix motif. The results suggest that synthetic amino acid alphabets with more strongly interacting side chains can be used to form polypeptides with improved binding properties in comparison to those engineered by biological methods.
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| 3. |
- T. Tegler, Lotta, et al.
(författare)
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Synthetic binder molecules that discriminate between isoforms of human Carbonic Anhydrase in blood.
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 4. |
- Tegler, Lotta T., et al.
(författare)
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Polypeptide Conjugate Binders that Discriminate between Two Isoforms of Human Carbonic Anhydrase in Human Blood
- 2011
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Ingår i: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 12:4, s. 559-566
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Tidskriftsartikel (refereegranskat)abstract
- Two binder candidates 4-C37L34-B and 3-C15L8-B from a 16-membered set of 42-residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence-based screening assay. Two carbonic anhydrase isoforms with 60% homology exist in human blood with HCAI being present in five-to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16-membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4-C37L34-B bound HCAII with a K-D of 17 nm and HCAI with a K-D of 470 nm, that is, with a 30-fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3-C15L8-B and the two carbonic anhydrases were 60 and 390 nm, respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.
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| 5. |
- Tegler, Lotta T., et al.
(författare)
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Powerful protein binders from designed polypeptides and small organic molecules : a general concept for protein recognition
- 2011
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 50:8, s. 1823-1827
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Tidskriftsartikel (refereegranskat)abstract
- High-affinity binders for the C-reactive protein (CRP), with dissociation constants in the pM to nM range and selectivities in human serum comparable to those of antibodies, were obtained by conjugation of 16 designed polypeptides to phosphocholine, a small molecule that binds CRP with a KDvalue of 5I . The polypeptides were not designed specifically to recognize CRP and bind by an adapted fit mechanism.
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