Identification of Specific Hydroxyapatite {001} Binding Heptapeptide by Phage Display and Its Nucleation Effect

Abstract

With recent developments of molecular biomimetics that combine genetic engineering and nanotechnology, peptides can be genetically engineered to bind specifically to inorganic components and execute the task of collagen matrix proteins. In this study, using biogenous tooth enamel as binding substrate, we identified a new heptapeptide (enamel high-affinity binding peptide, EHBP) from linear 7-mer peptide phage display library. Through the output/input affinity test, it was found that EHBP has the highest affinity to enamel with an output/input ratio of 14.814 × 10−7, while a random peptide (RP) displayed much lower output/input ratio of 0.00035 × 10−7. This binding affinity was also verified by confocal laser scanning microscopy (CLSM) analysis. It was found that EHBP absorbing onto the enamel surface exhibits highest normalized fluorescence intensity (5.6 ± 1.2), comparing to the intensity of EHBP to enamel longitudinal section (1.5 ± 0.9) (p < 0.05) as well as to the intensity of a low-affinity binding peptide (ELBP) to enamel (1.5 ± 0.5) (p < 0.05). Transmission electron microscopy (TEM), Attenuated total Reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and X-ray Diffraction (XRD) studies further confirmed that crystallized hydroxyapatite were precipitated in the mineralization solution containing EHBP. To better understand the nucleation effect of EHBP, EHBP was further investigated on its interaction with calcium phosphate clusters through in vitro mineralization model. The calcium and phosphate ion consumption as well as zeta potential survey revealed that EHBP might previously adsorb to phosphate (PO43−) groups and then initiate the precipitation of calcium and phosphate groups. This study not only proved the electrostatic interaction of phosphate group and the genetically engineering solid-binding peptide, but also provided a novel nucleation motif for potential applications in guided hard tissue biomineralization and regeneration.

Synthesis of Reduced Grapheme Oxide as A Platform for loading β-NaYF4:Ho3+@TiO2Based on An Advanced Visible Light-Driven Photocatalyst

“…7. The Raman spectrum of rGO displayed two prominent peaks at 1355.8 and 1582.2 cm −1 , corresponding to the well-documented D and G bands, respectively 65 . G band is Raman active for sp 2 hybridized carbon-based material, while D band is activated only if defects participate the double resonance Raman scattering near K point of Brillouin zone 63 .…”

Selection and identification of a novel bone-targeting peptide for biomedical imaging of bone

“…Therefore, we screened novel peptides with strong affinity and high specificity for binding hydroxyapatite from a randomized 8-mer peptide phage library using the methods of negative and positive selection. Hydroxyapatite-binding peptides screened from phage display or combinatorial peptide libraries have been reported previously [32][33][34] . However, most of these peptides have been used to control the nucleation and mineralization of hydroxyapatite formation.…”

Design of a hydroxyapatite-binding antimicrobial peptide with improved retention and antibacterial efficacy for oral pathogen control

“…This is a promising surrogate to silanisation and would not have a significant impact on the HAp surface. The HAp-binding domain of HBAMP was isolated from a linear 7-mer peptide phage display library, by using enamel as binding substrate20. The binding affinity of HBP7 with enamel was evaluated using Output/Input affinity test according to a standard protocol developed by the manufacture (New England Biolabs, Ipswich, MA, USA).…”

“…In addition, KSLW possesses merits in terms of stability in human saliva and safety in gastrointestinal tract, which are prerequisites for serving as a clinically used antimicrobial agent in oral cavity16. HAp-binding peptide (HBP7, NNHYLPR), isolated by bio-panning phage display random heptapeptide library, has exhibited specific affinity to enamel surface, making it a suitable molecule for tethering AMPs onto enamel surface20.…”