Development of a strategy to functionalize a dextrin-based hydrogel for animal cell cultures using a starch-binding module fused to RGD sequence.
BACKGROUND
Several approaches can be used to functionalize biomaterials, such as hydrogels, for biomedical applications. One of the molecules often used to improve cells adhesion is the peptide Arg-Gly-Asp (RGD). The RGD sequence, present in several proteins from the extra-cellular matrix (ECM), is a ligand for integrin-mediated cell adhesion; this sequence was recognized as a major functional group responsible for cellular adhesion. In this work a bi-functional recombinant protein, containing a starch binding module (SBM) and RGD sequence was used to functionalize a dextrin-based hydrogel. The SBM, which belongs to an alpha-amylase from Bacillus sp. TS-23, has starch (and dextrin, depolymerized starch) affinity, acting as a binding molecule to adsorb the RGD sequence to the hydrogel surface.


RESULTS
The recombinant proteins SBM and RGD-SBM were cloned, expressed, purified and tested in in vitro assays. The evaluation of cell attachment, spreading and proliferation on the dextrin-based hydrogel surface activated with recombinant proteins were performed using mouse embryo fibroblasts 3T3. A polystyrene cell culture plate was used as control. The results showed that the RGD-SBM recombinant protein improved, by more than 30%, the adhesion of fibroblasts to dextrin-based hydrogel. In fact, cell spreading on the hydrogel surface was observed only in the presence of the RGD-SBM.


CONCLUSIONS
The fusion protein RGD-SBM provides an efficient way to functionalize the dextrin-based hydrogel. Many proteins in nature that hold a RGD sequence are not cell adhesive, probably due to the conformation/accessibility of the peptide. We therefore emphasise the successful expression of a bi-functional protein with potential for different applications.
Moreira
Susana M
SM
IBB, Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal. smargarida@deb.uminho.pt
Andrade
Fábia K
FK
Domingues
Lucíla
L
Gama
Miguel
M
D016475:3T3 Cells; D000327:Adsorption; D000818:Animals; D002448:Cell Adhesion; D003001:Cloning, Molecular; D003912:Dextrins / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry*; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry*; D011137:Polystyrenes; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013213:Starch / Q000378:metabolism*
D003912:Dextrins; D020100:Hydrogels; D009842:Oligopeptides; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; C047981:arginyl-glycyl-aspartic acid; D013213:Starch