Ortsgerichtete Immobilisierung fluoreszenzmarkierter IL-6-Typ-Zytokine und Analyse ihrer Signaltransduktion

IL-6-type cytokines have a significant function in modulating the inflammatory response and wound healing process. They influence growth, proliferation and differentiation of various cell types. Therefore, the functionalization of biomaterial surfaces with IL-6-type cytokines permits the control of versatile interactions at the interface material/cell. Besides altering the biocompatibility of materials, immobilized cytokine may help to understand their signal transduction in more detail, e.g. allowing the precise analysis of cytokine/receptor-interaction. A requirement for such applications is the stable and site-specific coupling of the IL-6-type cytokine to prevent release or loss in bioactivity, which both would lead to misinterpreted results. The cytokines hLIF, hOSM and hIL-6 were fused at the N-terminus with different tags by use of molecular genetics methods. After expression of these recombinant cytokines in E. coli or HEK293-cells, these tags enabled a) the purification (His-tag), b) a reproducible, site-specific coupling in bioactive orientation (His-tag or SNAP-tag) on an adequately modified model-surface, c) the surface-detection (YFP). To obtain an efficient surface coupling, three site-specific immobilization strategies were analyzed:A) Coordinative bonding via the His-tag on TALON®-Matrix with cobalt-ionsB) Covalent coupling via the His-tag on a surface modified with phenylboronic acid C) Covalent coupling by the SNAP-tag on a surface modified with benzylguanine By use of the SNAP-tag method, the recombinant YFP-labled IL-6-type cytkines (hOSM and hIL-6) were permanently immobilized via their N-termini on a model surface (bead or microscopy-coverslip). The coupling of the fusion-cytokines was homogenous. A bioactive conformation was concluded because cytokine-modified beads were able to induce a STAT3 and an ERK1/2 phosphorylation. A normal translocation of STAT3 from the cytoplasm into the nucleus was shown by confocal laser scanning microscopy. Although the cytokines were immobilized, in comparison to soluble cytokines only slight differences in the temporal activation pattern of intracellular mediators were seen. This is most likely due to an unaffected expression of SOCS3 induced by STAT3.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2011

Author dc:creator

• Recker, Tobias

Contributors dc:contributor

• Müller-Newen, Gerhard

• info:eu-repo/classification/ddc/570
• Immobilisierung
• Cytokine
• Interleukin 6
• Signaltransduktion
• Biowissenschaften, Biologie
• Biofunktionalisierung
• SNAP-tag
• site-specific
• immobilization
• IL-6-type cytokine
• biofunctionalization
• signal transduction

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

ger

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-124479%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:63011