Purification of Recombinant Fusion Peptide Containing Hydroxyapatite Affinity Tag Using Ceramic Chromatography Column

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BAHRI M.; HASANNIA S.; DABIRMANESH B.; HOSEIN ZADEH H.

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Journal: JOURNAL OF BIOTECHNOLOGY Year:2019 | Volume:10 | Issue:4 Page(s): 653-662

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Title

Purification of Recombinant Fusion Peptide Containing Hydroxyapatite Affinity Tag Using Ceramic Chromatography Column

Author(s)

BAHRI M. | HASANNIA S. | DABIRMANESH B. | HOSEIN ZADEH H. | Issue Writer Certificate

Keywords

PeptideQ1

FusionQ2

HydroxyapatiteQ3

Heparin Binding DomainQ1

DLSQ1

Abstract

Introduction Nowadays, bone tissue repair with increasing bone disorders and injuries have special importance. Bone tissue engineering provided specific solutions to these problems. The present study was conducted with the aim of purification of recombinant Fusion Peptide containing Hydroxyapatite affinity tag using the ceramic chromatography column. Material & methods In this study, a Fusion Peptide was designed which at one side comprised the heparin-binding domain sequence, which can be attached to various types of growth factors involved in tissue repair and entrap these factors at the site of the lesion. On the other side, it contained a tag, which included a sequence derived from a laboratory study based on phage expression. The reason for keeping the sequence of this tag is to attach the Peptide to the scaffold containing Hydroxyapatite and purifying the recombinant Peptide by the Hydroxyapatite column. Therefore, the gene sequence was optimized and synthesized for expression in the prokaryotic host of E. coli strain BL21. Then the gene sequence was subcloned by double digestion with the SacI and BamHI enzymes into the expression vector of pET-21a(+). The expression of the recombinant Peptide was investigated by SDS-PAGE and western blot. In order to optimize the purification conditions, two-step purification was carried out by applying fundamental changes in the main work method of the manufacturer company and was purified with acceptable purity. Finally, the existence of Peptide assemblies was investigated by the SLD method. Finding The results of PCR cloning, enzymatic digestion using SacI and BamHI enzymes and sequencing indicated the accuracy of the cloning process. On the other hand, expression of the Fusion Peptide was confirmed by SDS-PAGE and Western blot techniques, and its migration onto the gel resulted in a band cleavage of about 12 kDa. Changes made to the manufacturer’ s workflow allowed the purification process to be optimized and the results of the DLS method showed the purity of the purified Peptide. Conclusion The results indicate the desirable expression and remarkable purity of the Fusion Peptide designed in this study.

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APA: Copy

BAHRI, M., HASANNIA, S., DABIRMANESH, B., & HOSEIN ZADEH, H.. (2019). Purification of Recombinant Fusion Peptide Containing Hydroxyapatite Affinity Tag Using Ceramic Chromatography Column. JOURNAL OF BIOTECHNOLOGY, 10(4 ), 653-662. SID. https://sid.ir/paper/231264/en

Vancouver: Copy

BAHRI M., HASANNIA S., DABIRMANESH B., HOSEIN ZADEH H.. Purification of Recombinant Fusion Peptide Containing Hydroxyapatite Affinity Tag Using Ceramic Chromatography Column. JOURNAL OF BIOTECHNOLOGY[Internet]. 2019;10(4 ):653-662. Available from: https://sid.ir/paper/231264/en

IEEE: Copy

M. BAHRI, S. HASANNIA, B. DABIRMANESH, and H. HOSEIN ZADEH, “Purification of Recombinant Fusion Peptide Containing Hydroxyapatite Affinity Tag Using Ceramic Chromatography Column,” JOURNAL OF BIOTECHNOLOGY, vol. 10, no. 4 , pp. 653–662, 2019, [Online]. Available: https://sid.ir/paper/231264/en

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