Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin.

Published

Journal Article

A dual ligand (DL) system that combines high affinity streptavidin-biotin binding with lower affinity fibronectin-integrin ligand binding was developed to augment endothelial cell adhesion to polymers. In this study, we examined the utility of biotinylated fibronectin (bFN) as an enhancement to the previously developed DL approach. The goal was to make the system more amenable to clinical studies by eliminating xenogenic bovine serum albumin (bBSA). Fibronectin (FN) biotinylation was achieved with Sulfo-NHS-LC-Biotin. The affinity of conjugated biotin for wild-type streptavidin (WT-SA) and a mutant strain streptavidin (RGD-SA) was measured using surface plasmon resonance (SPR) spectroscopy. Enzyme-Linked ImmunoSorbent Assay (ELISA) absorbance values confirmed the accessibility of the cell binding domain on mildly biotinylated bFN when compared to unmodified native protein. SPR binding analysis confirmed similar binding behavior to bFN with WT-SA and RGD-SA. Kinetic analysis, however, showed no increase in affinity due to increased biotins per FN, an indication of the absence of positive cooperativity in the system. We verified the essential utility of bFN in affinity binding by SPR and confirmed the potential for integrin-FN linkages by ELISA. Finally, Vinculin immunostaining was used to determine focal adhesion formation using bFN in the DL system. Significantly greater focal adhesion density was achieved with the bFN in the DL system than with FN alone.

Full Text

Duke Authors

Cited Authors

  • Anamelechi, CC; Clermont, EE; Brown, MA; Truskey, GA; Reichert, WM

Published Date

  • December 2007

Published In

Volume / Issue

  • 23 / 25

Start / End Page

  • 12583 - 12588

PubMed ID

  • 17985940

Pubmed Central ID

  • 17985940

Electronic International Standard Serial Number (EISSN)

  • 1520-5827

International Standard Serial Number (ISSN)

  • 0743-7463

Digital Object Identifier (DOI)

  • 10.1021/la702322n

Language

  • eng