Volume 15, Issue 57 (12-2024)
NCMBJ 2024, 15(57): 57-69 |
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Sadeghi-Ardebili M, Hasannia S, Dabirmanesh B, Khavari-Nejad R A. Dual application of hydroxyapatite tag in engineered PDGF peptide: purification and drug delivery. NCMBJ 2024; 15 (57) :57-69
URL: http://ncmbjpiau.ir/article-1-1718-en.html
URL: http://ncmbjpiau.ir/article-1-1718-en.html
Department of Biochemistry, Faculty of Biological Science, Tarbiat Modares University, Tehran,Iran , hasannia@modares.ac.ir
Abstract: (425 Views)
Aim and Background: The use of affinity tags is a conventional method for purifying recombinant proteins. However, their removal in later stages is mandatory for therapeutic peptides or proteins, which not only prolongs the purification process but also imposes significant costs on the producer. A peptide sequence with an affinity for hydroxyapatite allows for the purification of recombinant peptides or proteins while enabling their binding to hydroxyapatite-containing structures such as teeth, bones, and even bone grafts. This can play a significant role in biomaterials, tissue engineering, surgery, and dental implants. The goal of this study is to utilize affinity sequences that eliminate the need for removal after purifying the desired protein using affinity chromatography columns, which is a priority in biotechnology and therapeutics.
Material and Methods: In this study, a fusion peptide derived from platelet-derived growth factor (PDGF-BB) was designed, containing a hydroxyapatite-binding sequence and His6x tag. To remove the histidine tag, the sequence was designed with primers containing XhoI and NdeI restriction sites and was subsequently cloned into the pET21a (+) vector. Polymerase chain reaction (PCR) and double enzymatic digestion were performed. The designed fusion peptide was expressed in Escherichia coli, confirmed through SDS-PAGE electrophoresis, and verified using Western blotting. The recombinant fusion peptide was purified using hydroxyapatite resin. To evaluate peptide binding to the scaffold, a release test was conducted, which was assessed using the Bradford protein assay and curve analysis. Cellular viability, proliferation, and growth were examined via the MTT assay.
Results: Expression of the designed fusion peptide in Escherichia coli was optimized. Cloning in the pET21a(+) vector was confirmed after PCR and double enzymatic digestion through gene sequencing. The expression was validated by SDS-PAGE and Western blotting, showing a peptide band at approximately 17 kDa. The peptide release assay demonstrated that the PDGF-BB fusion peptide binds stably to the hydroxyapatite scaffold, preventing complete peptide release on the first day in SBF (Simulated Body Fluid). On the second day, approximately 40% of the peptide was released, followed by stabilization (plateau). The MTT assay revealed cell viability and proliferation over 24 hours, with maximum growth observed at a concentration of 2.9 µg/mL compared to the control group.
Conclusion: In this study, a novel peptide derived from PDGF-BB with stable performance was designed, expressed, and purified using hydroxyapatite resin. The dual-purpose use of the hydroxyapatite-binding affinity tag not only enhances its efficiency in purification processes using hydroxyapatite-based ceramic affinity columns but also serves as an affinity factor for bone scaffolds in tissue engineering.
Material and Methods: In this study, a fusion peptide derived from platelet-derived growth factor (PDGF-BB) was designed, containing a hydroxyapatite-binding sequence and His6x tag. To remove the histidine tag, the sequence was designed with primers containing XhoI and NdeI restriction sites and was subsequently cloned into the pET21a (+) vector. Polymerase chain reaction (PCR) and double enzymatic digestion were performed. The designed fusion peptide was expressed in Escherichia coli, confirmed through SDS-PAGE electrophoresis, and verified using Western blotting. The recombinant fusion peptide was purified using hydroxyapatite resin. To evaluate peptide binding to the scaffold, a release test was conducted, which was assessed using the Bradford protein assay and curve analysis. Cellular viability, proliferation, and growth were examined via the MTT assay.
Results: Expression of the designed fusion peptide in Escherichia coli was optimized. Cloning in the pET21a(+) vector was confirmed after PCR and double enzymatic digestion through gene sequencing. The expression was validated by SDS-PAGE and Western blotting, showing a peptide band at approximately 17 kDa. The peptide release assay demonstrated that the PDGF-BB fusion peptide binds stably to the hydroxyapatite scaffold, preventing complete peptide release on the first day in SBF (Simulated Body Fluid). On the second day, approximately 40% of the peptide was released, followed by stabilization (plateau). The MTT assay revealed cell viability and proliferation over 24 hours, with maximum growth observed at a concentration of 2.9 µg/mL compared to the control group.
Conclusion: In this study, a novel peptide derived from PDGF-BB with stable performance was designed, expressed, and purified using hydroxyapatite resin. The dual-purpose use of the hydroxyapatite-binding affinity tag not only enhances its efficiency in purification processes using hydroxyapatite-based ceramic affinity columns but also serves as an affinity factor for bone scaffolds in tissue engineering.
Keywords: Hydroxyl apatite, Protein purification, PDGF-BB, Platelet- derived growth factor, Tissue engineering, Wound healing
Type of Study: Research Article |
Subject:
Cellular and molecular
Received: 2025/02/26 | Accepted: 2024/12/21 | Published: 2024/12/21
Received: 2025/02/26 | Accepted: 2024/12/21 | Published: 2024/12/21
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