Kreiner, M., Li, Z., Beattie, J., Kelly, S. , Mardon, H. and van der Walle, C.F. (2008) Self-assembling multimeric integrin {alpha}5{beta}1 ligands for cell attachment and spreading. Protein Engineering Design and Selection, 21(9), pp. 553-560. (doi: 10.1093/protein/gzn032)
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Publisher's URL: http://dx.doi.org/10.1093/protein/gzn032
Abstract
Substrates utilising clustered arginine-glycine-aspartic acid (RGD) ligand displays support greater cell adhesion over random displays. However, cell adhesion to integrin {alpha}5{beta}1 requires the synergy site on the 9th type III fibronectin domain (FIII) in addition to RGD on the 10th FIII domain. Here, we have designed and expressed soluble protein chimeras consisting of an N-terminal 9th-10th FIII domain pair, IgG-derived hinge and leucine zipper-derived helix; the latter mutated to yield di-, tri- and tetrameric coiled coils and thus self-assembling, multimeric integrin {alpha}5{beta}1 ligands. A unique C-terminal cysteine was appended to the helix to facilitate anchoring' of the chimeras with a defined orientation on a surface. Size-exclusion chromatography and circular dichroism demonstrated that the chimeras self-assembled as multimers in solution with defined secondary structures predicted from theoretical calculations. Biotinylation via a thioether bond was used to selectively bind the chimeras to streptavidin-coated surfaces, each of which was then shown to bind integrin {alpha}5{beta}1 by surface plasmon resonance. Spreading of fibroblasts to surfaces derivatised with the chimeras was found to proceed in the order: tetramer > trimer > dimer > monomer. Thus, we describe novel polyvalent integrin {alpha}5{beta}1 ligands for facile derivatisation of substrates to improve cell adhesion in vitro
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Kelly, Dr Sharon |
Authors: | Kreiner, M., Li, Z., Beattie, J., Kelly, S., Mardon, H., and van der Walle, C.F. |
Subjects: | Q Science > QH Natural history > QH345 Biochemistry |
College/School: | College of Medical Veterinary and Life Sciences |
Journal Name: | Protein Engineering Design and Selection |
Journal Abbr.: | Peds |
ISSN: | 1741-0126 |
ISSN (Online): | 1741-0134 |
Published Online: | 30 May 2008 |
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