Partenaires et organisations internationales
(Anglais)
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Biotul, München (D), ENS Paris (F), VTT Tampere (FIN), Université Louvain-la-neuve (B)
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Résumé des résultats (Abstract)
(Anglais)
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The objective of BIOtul was the development of a novel, surface plasmon resonance based real-time biosensor device. This included the design of the instrument itself, the development of new hydrogels that allow the efficient coupling of biomolecules, and the testing and fine-tuning of the instrument using biomolecules.
Cytos task was the generation of biomolecules that allow real-life measurements on the SPR device. First, a highly ambitious project was started that aimed at the detection of translation errors in a biopharmaceutical product. These errors stem from the different DNA codon uses in humans and production organisms like E. coli. The misincorporation of amino acids might severely affect the specificity and efficacy of a biopharmaceutical drug and therefore a sensitive measurement device like the SPR based device that was the objective of this project would be of great value for such an analysis. The effect of codon usage on the translation of DNA into protein in E. coli can only be studied by comparison of the protein properties of two batches of protein that were either produced using the original, no optimized Human gene in E. coli or an optimized version, in which rare E. coli codons were exchanged. As a measurement for the specificity and efficacy of the two protein samples it was planned to detect the affinity of the biomolecule towards it natural receptor, as lower receptor binding strongly indicates reduced efficacy. As a real world model the human growth hormone was chosen, that is available as a commercial drug. In the first year of the project the two different expression plasmids coding for the human growth hormone were successfully constructed and preliminary expression tests were carried out.
In the second year the expression system for human growth hormone was optimized and a purification protocol was established, yielding protein pure enough for SPR measurements. However, the lack of a measurement device and problems during the construction phase required a change in strategy towards a well characterized model system, which offered the possibility to compare data obtained on the new device with the results obtained by using established instruments (the BIAcore). Therefore, a expression and purification process for a single chain antibody fragment (scFv) was developed. This scFv binds with a well characterized affinity to hen egg lysozyme, and comparative measurements on the BIAcore and the new SPR device would have allowed a fine tuning of device parameters. So a complete expression and purification protocol was developed, and protein material of sufficient quality for the above-mentioned experiments was obtained.
Finally, after the bankruptcy of BIOTul it was obvious the strategy had to be re-evaluated again. Therefore the partners agreed to focus on biomolecules the can be used at a very early development stage of the measurement device. First a system consisting of a synthetic peptide and an antibody recognizing this peptide with a high affinity was established. The peptide was coupled a via a free thiol group to free NH2 groups of a carrier protein using coupling chemistry that is used for the attachment of biomolecules to hydro gels. The recognition of the peptide by the antibody after coupling was verified for the denatured protein as well as in the native conformation. Second, it was agreed that a full-length protein with beneficial properties would be helpful for the evaluation of the development of hydro gels and establishing of the attachment of biomolecules to the hydro gels. The prerequisites for this protein were a certain stability, an efficient detection method and a functional test that allowed to judge whether a protein is still intact after coupling to hydro-gels. An additional requirement was the availability of a monoclonal antibody towards this biomolecule that would allow kinetic measurements on the SPR device. So two variants of the green fluorescent protein were constructed, one containing a free cysteine for covalent coupling to hydro gels via a free thiol group and the other a six-histidine tag for the non covalent attachment to a different type of hydro gel. Both protein variants could be produced in E. coli with good yields and were purified to a sufficient degree. In addition, for both variants the presence of the attachment site was verified.
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