Volume 10, Issue 4 (2019)
JMBS 2019, 10(4): 655-664 |
Back to browse issues page
Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:
Bahri M, Hasannia S, Dabirmanesh B, Zadeh H. Purification of Recombinant Fusion Peptide Containing Hydroxyapatite Affinity Tag Using Ceramic Chromatography Column. JMBS 2019; 10 (4) :655-664
URL: http://biot.modares.ac.ir/article-22-32148-en.html
URL: http://biot.modares.ac.ir/article-22-32148-en.html
1- Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran
2- Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran, Tarbiat Modares University, Nasr Bridge, Jalal-Al-Ahmad Highway, Tehran, Iran. Postal Code: 1411713116 ,hasannia@modares.ac.ir
3- Laboratory for Immunoregulation & Tissue Engineering, Dentistry Faculty, University of Southern California, Los Angeles, USA
2- Biochemistry Department, Biological Sciences Faculty, Tarbiat Modares University, Tehran, Iran, Tarbiat Modares University, Nasr Bridge, Jalal-Al-Ahmad Highway, Tehran, Iran. Postal Code: 1411713116 ,
3- Laboratory for Immunoregulation & Tissue Engineering, Dentistry Faculty, University of Southern California, Los Angeles, USA
Abstract: (4849 Views)
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.
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.
Article Type: Original Research |
Subject:
Molecular biotechnology
Received: 2019/04/20 | Accepted: 2019/07/24 | Published: 2019/12/21
Received: 2019/04/20 | Accepted: 2019/07/24 | Published: 2019/12/21
References
1. Bettinger CJ, Weinberg EJ, Kulig KM, Vacanti JP, Wang Y, Borenstein JT, et al. Three‐dimensional microfluidic tissue‐engineering scaffolds using a flexible biodegradable polymer. Adv Mater. 2006;18(2):165-9. [Link] [DOI:10.1002/adma.200500438]
2. Bessa PC, Casal M, Reis RL. Bone morphogenetic proteins in tissue engineering: The road from the laboratory to the clinic, part I (basic concepts). J Tissue Eng Regen Med. 2008;2(1):1-13. [Link] [DOI:10.1002/term.63]
3. Black CR, Goriainov V, Gibbs D, Kanczler J, Tare RS, Oreffo RO. Bone tissue engineering. Curr Mol Biol Rep. 2015;1(3):132-40. [Link] [DOI:10.1007/s40610-015-0022-2]
4. Tang D, Tare RS, Yang LY, Williams DF, Ou KL, Oreffo RO. Biofabrication of bone tissue: Approaches, challenges and translation for bone regeneration. Biomaterials. 2016;83:363-82. [Link] [DOI:10.1016/j.biomaterials.2016.01.024]
5. Malik MA, Puleo DA, Bizios R, Doremus RH. Osteoblasts on hydroxyapatite, alumina and bone surfaces in vitro; Morphology during the first 2h of attachment. Biomaterials. 1992;13(2):123-8. [Link] [DOI:10.1016/0142-9612(92)90008-C]
6. Pilipchuk SP, Plonka AB, Monje A, Taut AD, Lanis A, Kang B, et al. Tissue engineering for bone regeneration and osseointegration in the oral cavity. Dent Mater. 2015;31(4):317-38. [Link] [DOI:10.1016/j.dental.2015.01.006]
7. Martino MM, Briquez PS, Ranga A, Lutolf MP, Hubbell JA. Heparin-binding domain of fibrin (ogen) binds growth factors and promotes tissue repair when incorporated within a synthetic matrix. Proc Natl Acad Sci. 2013;110(12):4563-8. [Link] [DOI:10.1073/pnas.1221602110]
8. Rosa PAJ, Ferreira IF, Azevedo AM, Aires-Barros MR. Aqueous two-phase systems: A viable platform in the manufacturing of biopharmaceuticals. J Chromatogr A. 2010;1217(16):2296-305. [Link] [DOI:10.1016/j.chroma.2009.11.034]
9. Azevedo AM, Rosa PAJ, Ferreira IF, Raquel Aires-Barros M. Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. Trends Biotechnol. 2009;27(4):240-7. [Link] [DOI:10.1016/j.tibtech.2009.01.004]
10. Fields C, Li P, O'Mahony JJ, Lee GU. Advances in affinity ligand‐functionalized nanomaterials for biomagnetic separation. Biotechnol Bioeng. 2016;113(1):11-25. [Link] [DOI:10.1002/bit.25665]
11. Wood DW. New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol. 2014;26:54-61. [Link] [DOI:10.1016/j.sbi.2014.04.006]
12. Cummings LJ, Snyder MA, Brisack K. Protein chromatography on hydroxyapatite columns. In: Burgess RR, Deutscher MP, editors. Guide to protein purification. 2nd Edition. Cambridge: Academic Press; 2009. [Link] [DOI:10.1016/S0076-6879(09)63024-X]
13. Snyder MA. Working with a powerful and robust mixed-mode resin for protein purification. BioProcess Int. 2011;9(5):50-53. [Link]
14. Roy MD, Stanley SK, Amis EJ, Becker ML. Identification of a highly specific hydroxyapatite‐binding peptide using phage display. Adv Mater. 2008;20(10):1830-6. [Link] [DOI:10.1002/adma.200702322]
15. He F. Laemmli-sds-page. Bio-protocol. 2011;1(11):e80. [Link] [DOI:10.21769/BioProtoc.80]
16. He F. Bradford protein assay. Bio-protocol. 2011;1(6):e45. [Link] [DOI:10.21769/BioProtoc.45]
17. Gagnon P, Frost R, Tunón P, Ogawa T. CHT™ ceramic hydroxyapatite: A new dimension in chromatography of biological molecules [Internet]. Hercules: Bio-Rad Laboratories; 1996 [cited 2018 October 23]. Available from: https://www.bio-rad.com/webroot/web/pdf/psd/literature/Bulletin_2156.pdf [Link]
18. Kawasaki T. Theory of chromatography on hydroxyapatite columns with small loads: V. determination of the adsorption energy of the ε-amino group of poly-l-lysine and the manner of adsorption of the molecule. J Chromatogr A. 1978;157:7-42. [Link] [DOI:10.1016/S0021-9673(00)92319-7]
19. Kawasaki T. Hydroxyapatite as a liquid chromatographic packing. J Chromatogr A. 1991;544:147-84. [Link] [DOI:10.1016/S0021-9673(01)83984-4]
20. Fernandez-Yague MA, Abbah SA, McNamara L, Zeugolis DI, Pandit A, Biggs MJ. Biomimetic approaches in bone tissue engineering: Integrating biological and physicomechanical strategies. Adv Drug Deliv Rev. 2015;84:1-29. [Link] [DOI:10.1016/j.addr.2014.09.005]
21. Leach JK, Mooney DJ. Bone engineering by controlled delivery of osteoinductive molecules and cells. Expert Opin Biol Ther. 2004;4(7):1015-27. [Link] [DOI:10.1517/14712598.4.7.1015]
22. Santo VE, Gomes ME, Mano JF, Reis RL. Controlled release strategies for bone, cartilage, and osteochondral engineering-Part II: Challenges on the evolution from single to multiple bioactive factor delivery. Tissue Eng Part B Rev. 2013;19(4):327-52. [Link] [DOI:10.1089/ten.teb.2012.0727]
23. Suárez-González D, Lee JS, Diggs A, Lu Y, Nemke B, Markel M, et al. Controlled multiple growth factor delivery from bone tissue engineering scaffolds via designed affinity. Tissue Eng Part A. 2013;20(15-16):2077-87. [Link] [DOI:10.1089/ten.tea.2013.0358]
24. Mouriño V, Cattalini JP, Roether JA, Dubey P, Roy I, Boccaccini AR. Composite polymer-bioceramic scaffolds with drug delivery capability for bone tissue engineering. Expert Opin Drug Deliv. 2013;10(10):1353-65. [Link] [DOI:10.1517/17425247.2013.808183]
25. Zhang H, Migneco F, Lin CY, Hollister SJ. Chemically-conjugated bone morphogenetic protein-2 on three-dimensional polycaprolactone scaffolds stimulates osteogenic activity in bone marrow stromal cells. Tissue Eng Part A. 2010;16(11):3441-8. [Link] [DOI:10.1089/ten.tea.2010.0132]
26. Islam T, Aguilar-Yañez JM, Simental-Martínez J, Ortiz-Alcaraz CI, Rito-Palomares M, Fernandez-Lahore M. A novel strategy for the purification of a recombinant protein using ceramic fluorapatite-binding peptides as affinity tags. J Chromatogr A. 2014;1339:26-33. [Link] [DOI:10.1016/j.chroma.2014.02.079]
27. Walls D, Loughran ST. Tagging recombinant proteins to enhance solubility and aid purification. In: Walls D, Sinéad D, Loughran ST, editors. Protein chromatography: Methods and protocols. Totowa: Humana Press; 2011. pp. 151-75. [Link] [DOI:10.1007/978-1-60761-913-0_9]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |