Binding and orientation of fibronectin to silanated glass surfaces using immobilized bacterial adhesin-related peptides.
Previously, we have demonstrated the suitability of bacterial adhesin-related peptides, directly immobilized on polystyrene surfaces, to bind and orient fibronectin (FN). For these studies a method to bind the large protein FN in a desired orientation on a solid substratum was developed which utilizes a bacterial adhesin-related peptide (designated BRP-A), which is known to bind specifically to the NH3-terminus end of FN. Glass substrata was first coated with an amine-terminated silane, followed by streptavidin (SA), which was used as an intermediate tether to bind the biotinylated bacterial adhesin-related peptide. The BRP-A peptide, used for these studies was synthesized with a terminal biotin to assure irreversible coupling of the BRP-A to the streptavidin. The biotinylated BRP-A was next immobilized on the SA-silanated glass surfaces. 125I-FN was used to quantify the amount of FN binding to the (BRP-A):SA-silanated glass surface. Monoclonal antibodies, which react with specific epitopes at either the NH3- or -COOH-termini of FN, were used to quantify the binding and orientation of FN. The results of these studies indicated: (1) FN bound to the BRP-A:SA-silanated glass surface; and (2) the bound FN was oriented such that NH2-terminal region of FN was bound towards the glass surface and the COOH-terminus was oriented away from the glass surface. These studies demonstrate that small peptides can be used to specifically bind and orient large proteins such as FN on the surfaces.
Klueh
U
U
The Center for Biomaterials, University of Connecticut, School of Dental Medicine, 263 Farmington, Avenue, Farmington, CT 06030-3105, USA.
Seery
T
T
Castner
D G
DG
Bryers
J D
JD
Kreutzer
D L
DL
D018829:Adhesins, Bacterial / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D005898:Glass / Q000737:chemistry; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012821:Silanes / Q000737:chemistry; D013052:Spectrometry, X-Ray Emission; D019809:Streptavidin / Q000737:chemistry
D018829:Adhesins, Bacterial; D005353:Fibronectins; D010455:Peptides; D012821:Silanes; D019809:Streptavidin