| 1. |
- Ramapanicker, Ramesh, et al.
(författare)
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Labelling of a polypeptide conjugate binder for the C-reactive protein with 68Ga for PET imaging
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- A polypeptide conjugate that binds the C-reactive protein (CRP) with high affinity and specificity in human serum has been labelled with the short-lived radionuclide 68Ga (t1/2 = 68 min) for PET imaging. The binder molecule was formed by conjugating phosphocholine to the side chain of a lysine residue of a 42-residue designed polypeptide via an aliphatic spacer. For radiolabelling, a DOTA group was covalently attached to the polypeptide via a PEG spacer linked to the side chain of a Cys residue. The affinity of the binder for CRP is due to the interactions between CRP and the phosphocholine group as well as between CRP and the polypeptide scaffold itself. The affinity for CRP of the polypeptide conjugate was not affected by the incorporation of the DOTA and the dissociation constant was shown by fluorescence titration to be in the low nM range. Life times of polypeptide conjugates in human serum have been shown to depend on the level of incorporation of residues that are not recognized by proteases and thus to be tunable by design. The apparent half life of the polypeptide scaffold without conjugated groups was 10–20 min at 37 °C in human serum whereas the half-life of the labelled CRP binder was more than 24 h under the same conditions. The molecular versatility of peptides and peptide conjugates has therefore been shown to make them excellent carriers of multiple functions for in vivo applications, as illustrated here by the combination of molecular recognition with short-lived radioisotopes for PET imaging.
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| 2. |
- Ramapanicker, Ramesh, et al.
(författare)
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Powerful binders for the D-dimer by conjugation of the GPRP peptide to polypeptides from a designed set : illustrating a general route to new binders for proteins
- 2013
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 24:1, s. 17-25
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Tidskriftsartikel (refereegranskat)abstract
- The synthetic tetrapeptide GPRP based on the amino-terminal GPR sequence of the fibrin α-chain, binds the D-dimer protein with a dissociation constant KD of 25 μM. The D-dimer protein, a well-known biomarker for thrombosis, contains two cross-linked D fragments from the fibrinogen protein formed upon degradation of the fibrin gel, the core component of blood clots. In order to develop a specific high-affinity binder for the D-dimer protein, GPRP was conjugated via an aliphatic spacer to each member of a set of sixteen polypeptides designed for the development of binder molecules for proteins in general. The binders were individually characterised and ranked using surface plasmon resonance (SPR) analysis. The dissociation constant of the complex formed from the D-dimer and 4-D15L8-GPRP labelled with fluorescein was determined by fluorescense titration and found to be 3 nM, an affinity four orders of magnitude higher than that of free GPRP. According to SPR analysis binding was completely inhibited by free GPRP at mM concentrations and the polypeptide conjugate was therefore shown to bind specifically to the binding site of GPRP. Affinities were further enhanced by dimerisation of the polypeptide conjugates via a bifunctional linker resulting in dissociation constants that were further decreased (affinities increased) by factors of 2-4. The results suggest an efficient route to specific binders for proteins based on short peptides with affinites that need only to be modest, thus shortening the time of binder development dramatically.
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| 3. |
- Ślósarczyk, Adam T., 1983-, et al.
(författare)
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Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids : efficient tools for peptide and protein conjugation
- 2012
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Ingår i: RSC Advances. - 2046-2069. ; 2:3, s. 908-914
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Tidskriftsartikel (refereegranskat)abstract
- An efficient methodology for the heteroconjugation of biomolecules with exposed free amino groups has been developed. Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids with aliphatic chains of varying sizes have been prepared and used to conjugate a 42-residue polypeptides with short model peptides as well as a model dodecapeptide with the antigenic determinant of type B blood, a carbohydrate derivative, to form a glycopeptide. The concept is based on the difference in reactivity towards primary amino groups between phenyl esters with leaving groups of unlike pKa. The reactivities of several pentafluorophenyl and o-fluorophenyl esters towards amino groups were carefully determined under reaction conditions to identify leaving group combinations that would provide optimal differences in reactivity for maximum yields of heteroconjugate formation while keeping the reasonable reaction times. Pentafluorophenyl esters react faster with an amino group and require a weaker base, while an o-fluorophenyl ester requires a stronger base and longer reaction time. The method described is economic, quick and gives complete control over the conjugation reaction. The size of the spacer is conveniently varied by selection of the appropriate aliphatic dicarboxylic acid. While the presented examples describe conjugation reactions of polypeptides with a maximum of 42 residues it is envisioned that the bifunctional linkers reported here will find their most important applications in the heteroconjugation of proteins using lysine side chains, a reaction for which currently few alternatives exist, if access to spacers of variable size is required.
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| 4. |
- 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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| 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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