DNA Hybridization-Based Differential Peptide Display Identified Potential Osteogenic Peptides
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DNA Hybridization‑Based Differential Peptide Display Identified Potential Osteogenic Peptides Wataru Hatakeyama1,2 · Cliff Lee1 · John Da Silva3 · David M. Kim1 · Shigemi Nagai1 · Hisatomo Kondo2 · Masazumi Nagai1 Accepted: 6 November 2020 © The Author(s) 2020
Abstract A DNA hybridization-based differential peptide display (DPD) was developed for the screening of phage peptide library to find osteogenic peptides intended to bind to epigenetically induced osteogenic receptors on NIH/3T3 (3T3) cell surface. In the presence of DNA methylation inhibitor of 5-azacytidine (5AZC), an osteoblastic receptor of bone morphogenetic protein (BMP) receptor 1A (BMPR1A) was induced on the cell surface of NIH/3T3 fibroblasts. Cyclic heptamer-displaying phage library was screened against vehicle and 5AZC treated (Tx) 3T3 cells. Antisense oligo against library against library peptide coding DNA of control 3T3 cell bound phages were synthesized to subtract common binders from that of 5AZC-Tx 3T3 cell-bound phages that included 5AZC-induced receptor binders. The library peptide coding regions of conformational receptor binder-subtracted DPD were PCR-amplified and cloned into a plasmid vector specifically designed for short peptide expression. No unique binder was identified when 96 clones were randomly picked from the third round of panning against 5AZC-treated 3T3 cells, suggesting miscellaneous bindings to cell surface proteins. Unique binders showing homology to known function proteins were successfully identified when constitutive receptor binders were subtracted from 5AZC-induced protein binders. Some of identified peptides significantly increased alkaline phosphatase activity in 5AZC-Tx 3T3 cells. DPD can be a useful tool to screen functional peptide bindings to cell surface receptors.
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13
Vol.:(0123456789)
International Journal of Peptide Research and Therapeutics
Graphic Abstract
Keywords Osteogenic peptide · Phage display · Whole cell panning · Differential subtraction · DNA hybridization
Introduction Iliac crest autogenous bone grafts have long served as the gold standard for the restoration of large bone defects, such as in surgical excision sites of bone metastatic tumors and alveolar cleft palate and traumatic bone loss (Giannoudis and Dinopoulos 2005). However, donor site morbidity and complications due to graft harvesting are common, including postoperative and chronic pain, infection, pelvic instability, scar formation and contour defects, and nerve injury and sensory disturbances (Kalk et al. 1996; Banwart et al. 1995; Myeroff and Archdeacon 2011; Canady et al. 1993). Although autogenous bone is well regarded for its self-healing abilities and has high success in treatment, its use is limited by the amount of autograft that can be obtained, as well shaping the graft in complex defects (Salgado et al. 2004). Alternatives to autologous bone grafts include allograft bone, metal, and ceramic plates/implants and scaffolds, including custom 3D
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