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Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin.

Publication ,  Journal Article
Anamelechi, CC; Clermont, EE; Brown, MA; Truskey, GA; Reichert, WM
Published in: Langmuir : the ACS journal of surfaces and colloids
December 2007

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.

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Published In

Langmuir : the ACS journal of surfaces and colloids

DOI

EISSN

1520-5827

ISSN

0743-7463

Publication Date

December 2007

Volume

23

Issue

25

Start / End Page

12583 / 12588

Related Subject Headings

  • Vinculin
  • Surface Plasmon Resonance
  • Streptavidin
  • Serum Albumin, Bovine
  • Ligands
  • Humans
  • Fibronectins
  • Enzyme-Linked Immunosorbent Assay
  • Endothelial Cells
  • Chemical Physics
 

Citation

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Anamelechi, C. C., Clermont, E. E., Brown, M. A., Truskey, G. A., & Reichert, W. M. (2007). Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin. Langmuir : The ACS Journal of Surfaces and Colloids, 23(25), 12583–12588. https://doi.org/10.1021/la702322n
Anamelechi, Charles C., Edward E. Clermont, Melissa A. Brown, George A. Truskey, and William M. Reichert. “Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin.Langmuir : The ACS Journal of Surfaces and Colloids 23, no. 25 (December 2007): 12583–88. https://doi.org/10.1021/la702322n.
Anamelechi CC, Clermont EE, Brown MA, Truskey GA, Reichert WM. Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin. Langmuir : the ACS journal of surfaces and colloids. 2007 Dec;23(25):12583–8.
Anamelechi, Charles C., et al. “Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin.Langmuir : The ACS Journal of Surfaces and Colloids, vol. 23, no. 25, Dec. 2007, pp. 12583–88. Epmc, doi:10.1021/la702322n.
Anamelechi CC, Clermont EE, Brown MA, Truskey GA, Reichert WM. Streptavidin binding and endothelial cell adhesion to biotinylated fibronectin. Langmuir : the ACS journal of surfaces and colloids. 2007 Dec;23(25):12583–12588.
Journal cover image

Published In

Langmuir : the ACS journal of surfaces and colloids

DOI

EISSN

1520-5827

ISSN

0743-7463

Publication Date

December 2007

Volume

23

Issue

25

Start / End Page

12583 / 12588

Related Subject Headings

  • Vinculin
  • Surface Plasmon Resonance
  • Streptavidin
  • Serum Albumin, Bovine
  • Ligands
  • Humans
  • Fibronectins
  • Enzyme-Linked Immunosorbent Assay
  • Endothelial Cells
  • Chemical Physics