Volume 24, Issue 3 (Jun 2016)                   JSSU 2016, 24(3): 232-240 | Back to browse issues page

XML Persian Abstract Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Moosavizadeh S, zeinodini M, Saeeidinia A, Nasiri Khlili M. Cloning and Expression of Ontak Immunotoxin Using Intein Tag. JSSU. 2016; 24 (3) :232-240
URL: http://jssu.ssu.ac.ir/article-1-3555-en.html
Abstract:   (5522 Views)

Introduction: Inteins (INT) are internal parts of a number of proteins in yeast and some other unicellular eukaryotes, which can be separated from the immature protein during protein splicing process. After identifying the mechanism of intein action, applications of these sequences are be considered in the single- step purification of recombinant proteins and different intein tags were developed. The most important advantage of using intein tags in purification of recombinant proteins than other affinity tags is no requirement of expensive protease enzymes and following additional steps to remove protease that make intein tags economically are considered more important. In the present study, denileukin diftitox immunotoxin (brand name Ontak), be fused with an intein tag and it was inserted in pTXB1 plasmid.

Methods: In this study, with respect to multiple cloning sites (MCS) of pTXB1, specific primers were designed. Polymerase Chain Reaction (PCR) was performed and encoding sequence of ONTAK was cloned using restriction sites of NdeI and SapI. Recombinant vector (PTX-IDZ) was transformed into E. coli strain ER2566 and expression of gene was studied.

Results: The accuracy of recombinant construct was confirmed by PCR and enzymatic digestion. The produced recombinant proteins were confirmed by SDS-PAGE and Western blotting.

Conclusion: Restriction site of SapI guarantees no additional residues incorporate in primary protein sequence. Also, the expression of this construct was analyzed in compare with fused protein to poly-His tag. According to the appropriate expression of fused protein in both constructs it was expected that one step- purification of considered drug protein will be success in the following steps.

Full-Text [PDF 1262 kb]   (1691 Downloads)    
Type of Study: Original article | Subject: Genetics
Received: 2016/01/6 | Accepted: 2016/04/9 | Published: 2016/07/25

1. Elleuche S, Pöggeler S. Inteins, valuable genetic elements in molecular biology and biotechnology. App microbio biotechno 2010; 87(2): 479-89.
2. Chong S, Shao Y, Paulus H, Benner J, Perler FB, Xu M-Q. Protein Splicing Involving the Saccharomyces cerevisiae VMA Intein The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J Biological Chemistry 1996; 271(36): 22159-68.
3. Klabunde T, Sharma S, Telenti A, Jacobs WR, Sacchettini JC. Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing. Nature Structural & Molecular Biology 1998; 5(1): 31-6.
4. Wood DW, Camarero JA. Intein applications: from protein purification and labeling to metabolic control methods. J Biologic Chemistry 2014; 289(21): 14512-519.
5. Chong S, Mersha FB, Comb DG, Scott ME, Landry D, Vence LM, et al. Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 1997; 192(2): 271-81.
6. Chong S, Montello G, Zhang A, Cantor EJ, Liao W, Xu M-Q, et al. Utilizing the C-terminal cleavage activity of a protein splicing element to purify recombinant proteins in a single chromatographic step. Nucleic acids Res 1998; 26(22): 5109-15.
7. Southworth MW, Amaya K, Evans TC, Xu M-Q, Perler FB. Purification of proteins fused to either the amino or carboxy terminus of the Mycobacterium xenopi gyrase A intein. Biotechniques 1999; 27(1): 110-04.
8. Li Y. Self-cleaving fusion tags for recombinant protein production. Biotechno letters 2011; 33(5): 869-81.
9. 9- Kreitman RJ. Immunotoxins for targeted cancer therapy. The AAPS J 2006; 8(3): E532-E51.
10. Frankel AE, Kreitman RJ, Sausville EA. Targeted toxins. Clinic cancer Res 2000; 6(2): 326-34.
11. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin treatment of cancer*. Annu Rev Med 2007; 58: 221-37.
12. Pastan I, Hassan R, FitzGerald DJ, Kreitman RJ. Immunotoxin therapy of cancer. Nature Reviews Cancer. 2006; 6(7): 559-65.
13. Kaminetzky D, Hymes KB. Denileukin diftitox for the treatment of cutaneous T-cell lymphoma. Biologics: Targets & Therapy 2008; 2(4): 717-24.
14. Knobler E. Current management strategies for cutaneous T-Cell Lymphoma. Clinics in dermatology 2004; 22(3): 197-208.
15. Amraee M, Zeinodini M, Soleimani M, Saeedinia AR. Cloning, expression, purification and toxicity assessment of diphtheria toxin - interleukin 2 fusion protein. J Polic Med 2015; (2): 125-32. [Persian]
16. Armstrong AW, Golan DE, Tashjian AH, Armstrong EJ, Armstrong AW. Principles of pharmacology: the pathophysiologic basis of drug therapy. 3rd edition, Wolters Kluwer Health/Lippincott Williams Wilkins, Philadelphia, 2008.
17. Mahmood I, Green MD. Pharmacokinetic and pharmacodynamic considerations in the development of therapeutic proteins. Clinic pharmacokinetics 2005; 44(4): 331-47.
18. Turturro F. Denileukin diftitox: a biotherapeutic paradigm shift in the treatment of lymphoid-derived disorders. Expert review anticancer therapy 2007; 7(1): 11-7.
19. Putney SD, Burke PA. Improving protein therapeutics with sustained-release formulations. Nature biotechno 1998; 16(2): 153-57.
20. Ballantyne A, Dhillon S. Trastuzumab emtansine: first global approval. Drugs 2013; 73(7): 755-65.
21. Cunningham D, Humblet Y, Siena S, Khayat D, Bleiberg H, Santoro A, et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer. New England J Med 2004; 351(4): 337-45.
22. Gogarten JP, Senejani AG, Zhaxybayeva O, Olendzenski L, Hilario E. Inteins: structure, function, and evolution. Annual Rev Microbio 2002; 56(1): 263-87.
23. Elleuche S, Döring K, Pöggeler S. Minimization of a eukaryotic mini-intein. Biochemical and biophysical Res commu 2008; 366(1): 239-43.
24. Naumann TA, Savinov SN, Benkovic SJ. Engineering an affinity tag for genetically encoded cyclic peptides. Biotechnology and bioengineering 2005; 92(7): 820-30.
25. Tavassoli A, Benkovic SJ. Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli. Nature protocols 2007; 2(5): 1126-33.
26. Banki MR, Gerngross TU, Wood DW. Novel and economical purification of recombinant proteins: Intein‐mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association. Protein Sci 2005; 14(6): 1387-95.
27. Shi C, Meng Q, Wood D. A dual ELP-tagged split intein system for non-chromatographic recombinant protein purification. App Microbio Biotechno 2013; 97(2): 829-35

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2022 CC BY-NC 4.0 | SSU_Journals

Designed & Developed by : Yektaweb