Volume 10, Issue 3 (2019)                   JMBS 2019, 10(3): 473-481 | Back to browse issues page

XML Persian Abstract Print

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

Askari S, Hasannia S, Hasan Sajedi R, Yassaee V. Cloning, Expression, and Purification of Recombinant CEL I Endonuclease in HEK293T Cell Line. JMBS 2019; 10 (3) :473-481
URL: http://biot.modares.ac.ir/article-22-26538-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. , hasannia@modares.ac.ir
3- “Genomic Research Center” and “Medical Genetics Department, Madicine Faculty”, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract:   (3549 Views)
CEL I endonuclease pertaining to the S1 endonuclease family. The enzyme, with its high specificity, has the ability to identify different types of mutations and base replacement in the DNA molecule, which makes it important in commercial products to use in research and clinical laboratories. Although the enzyme exists in the celery plant, the extraction of the enzyme is a time-consuming process and not economical and the yield of the final product is low. In addition, due to its post-translational modifications to achieve the final active structure, no report has published to indicate the expression of the active form of this enzyme in the bacterial hosts yet. Therefore, one of the production sources of the active form of this enzyme is its cloning and expression in eukaryotic hosts, including yeast and mammalian cell lines. In this study, in order to express CEL I endonuclease, its gene sequence was optimized and synthesized in host eukaryotic HEK293T. CEL I was subcloned by double digest with KpnI and XhoI enzymes in the pBudCE4.1expression vector. The expression construct was transfected into the HEK293T cell line by lipofectamine transfection. Expression of the recombinant protein after transfection into HEK293T cells was confirmed by multiple methods including polyacrylamide gel electrophoresis, ELISA, RT-PCR, and western blot reaction. The analysis of SDS-PAGE and western blot data confirmed the molecular weight of approximately 30kDa. Purification was carried out with the Ni-NTA column and the amount of purified protein was determined to be about 0.2mg/ml. Finally, the activity of endonuclease enzyme was investigated on both normal and mutated heteroduplex DNA amplified by PCR. The results showed that the expression of this protein in HEK293T host had shown sufficient activity.
Full-Text [PDF 1229 kb]   (1791 Downloads)    
Article Type: Original Research | Subject: Molecular biotechnology
Received: 2018/10/27 | Accepted: 2018/12/15 | Published: 2019/09/21

1. Oleykowski CA, Bronson Mullins CR, Godwin AK, Yeung AT. Mutation detection using a novel plant endonuclease. Nucleic Acids Res. 1998;26(20):4597-602 [Link] [DOI:10.1093/nar/26.20.4597]
2. Yang B, Wen X, Kodali NS, Oleykowski CA, Miller CG, Kulinski J, et al. Purification, cloning, and characterization of the CEL I nuclease. Biochemistry. 2000;39(13):3533-41. [Link] [DOI:10.1021/bi992376z]
3. Sabetta W, Alba V, Blanco A. sunTILL: A TILLING resource for gene function analysis in sunflower. Plant methods, 2011;7(1):20. [Link] [DOI:10.1186/1746-4811-7-20]
4. Colbert T, Till BJ, Tompa R, Reynolds S, Steine MN, Yeung AT, Henikoff S. High-throughput screening for induced point mutations. Plant Physiol. 2001;126(2):480-4. [Link] [DOI:10.1104/pp.126.2.480]
5. McCallum CM, Comai L, Greene EA, Henikoff S. Targeting induced locallesions in genomes (TILLING) for plant functional genomics. Plant Physiol. 2000;123(2):439-42. [Link] [DOI:10.1104/pp.123.2.439]
6. Perry JA, Wang TL, Welham TJ, Gardner S, Pike JM, Yoshida S, Parniske M. A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol. 2003;131(3):866-71. [Link] [DOI:10.1104/pp.102.017384]
7. Raghavan C, Naredo MEB, Wang H, Atienza G, Liu B, Qiu F, Leung H. Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping. Mol Breed. 2007;19(2):87-101. [Link] [DOI:10.1007/s11032-006-9046-x]
8. Till BJ, Burtner C, Comai L, Henikoff S. Mismatch cleavage by single‐strand specific nucleases. Nucleic Acids Res. 2004;32(8):2632-41. [Link] [DOI:10.1093/nar/gkh599]
9. Yeung AT, Hattangadi D, Blakesley L, Nicolas E. Enzymatic mutation detection technologies. Biotechniques. 2005;38(5):749-58. [Link] [DOI:10.2144/05385RV01]
10. Pimkin M, Caretti E, Canutescu A, Yeung JB, Cohn H, Chen Y, et al. Recombinant nucleases CEL I from celery and SP I from spinach for mutation detection. BMC Biotechnol. 2007;7:29. [Link] [DOI:10.1186/1472-6750-7-29]
11. Mon H, Lee J, Fukushima M, Nagata Y, Fujii M, Xu J, et al. Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system. Appl Microbiol Biotechnol. 2013;97(15):6813-22. [Link] [DOI:10.1007/s00253-012-4583-1]
12. Desai NA, Shankar V. Single-strand-specific nucleases. FEMS Microbiol Rev. 2003;26(5):457-91. [Link] [DOI:10.1111/j.1574-6976.2003.tb00626.x]
13. Triques K, Sturbois B, Gallais S, Dalmais M, Chauvin S, Clepet C, et al. Characterization of arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. Plant J. 2007;51(6):1116-25. [Link] [DOI:10.1111/j.1365-313X.2007.03201.x]
14. Bendahmane A, Sturbois B, Triques K, Caboche M. Method for producing highly sensitive endonucleases, novel preparations of endonucleases and uses thereof U.S. Patent Appl. 2012;13/183,565. [Link]
15. Bendahmane A, Sturbois B, Triques K, Caboche M. Method for producing highly sensitive endonucleases, novel preparations of endonucleases and uses thereof. U.S. Patent Appl. 2012;11/572,860. [Link]
16. Diepenbruck C, Klinger M, Urbig T, Baeuerle P, Neef R. Productivity and quality of recombinant proteins produced by stable CHO cell clones can be predicted by transient expression in HEK cells. Mol Biotechnol. 2013;54(2):497-503. [Link] [DOI:10.1007/s12033-012-9590-z]
17. Wurm F, Bernard A. Large-scale transient expression in mammalian cells for recombinant protein production. Curr Opin Biotechnol. 1999;10(2):156-9. [Link] [DOI:10.1016/S0958-1669(99)80027-5]
18. Hacker DL, Kiseljak D, Rajendra Y, Thurnheer S, Baldi L, Wurm FM. Polyethyleneimine-based transient gene expression processes for suspension-adapted HEK-293E and CHO-DG44 cells. Protein Expr Purif. 2013;92(1):67-76. [Link] [DOI:10.1016/j.pep.2013.09.001]
19. Qiu P, Shandilya H, D'Alessio JM, O'Connor K, Durocher J, Gerard GF. Mutation detection using Surveyor™ nuclease. Biotechniques. 2004;36(4):702-7. [Link] [DOI:10.2144/04364PF01]
20. He F. 23 users have reported that they have successfully carried out the experiment using this protocol. Laemmli-SDS-PAGE [Internet]. Sunnyvale, CA: Bio-protocol; 2011 [cited 2018 May 15]. Available from: https://bio-protocol.org/bio101/e80. [Link]
21. He F. Bradford protein assay [Internet]. Sunnyvale, CA: Bio-protocol; 2011 [cited 2018 May 20]. Available from: https://bio-protocol.org/bio101/e45 [Link]
22. Jahani Z, Mollakarimi M, Hasan Sajedi R, Taghdir M, Hosseinkhani S, Asghari SM. The role of some amino acid residues in coelenterazine binding site of mnemiopsin compared to coelenterate photoproteins. Modares J Biotechnol. 2014;5(1):30-40. [Persian] [Link]
23. Abdullaev SA, Gulyaeva NA, Bezlepkin VG, Gaziev AI. Comparative assessment of mutations in mtDNA of x-irradiated mice using mismatch-specific endonuclease and temporal temperature gradient electrophoresis. Biophysics. 2010;55(1);142-7. [Link] [DOI:10.1134/S0006350910010215]
24. Bronner CE, Baker SM, Morrison PT, Warren G, Smith LG, Lescoe MK, et al. Mutation in the DNA mismatch repair gene homologue hMLH 1 is associated with hereditary non-polyposis colon cancer. Nature. 1994;368(6468):258-61. [Link] [DOI:10.1038/368258a0]
25. Friedman LS, Ostermeyer EA, Szabo CI, Dowd P, Lynch ED, Rowell SE, King MC. Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nat Genet. 1994;8(4):399-404. [Link] [DOI:10.1038/ng1294-399]
26. Ito M, Oiso Y, Murase T, Kondo K, Saito H, Chinzei T, Racchi M, Lively MO. Possible involvement of inefficient cleavage of preprovasopressin by signal peptidase as a cause for familial central diabetes insipidus. J Clin Invest. 1993;91(6):2565-71. [Link] [DOI:10.1172/JCI116494]
27. Nicolaides NC, Papadopoulos N, Liu B, Weit YF, Carter KC, Ruben SM, et al. Mutations of two P/WS homologues in hereditary nonpolyposis colon cancer. Nature. 1994;371(6492):75-80. [Link] [DOI:10.1038/371075a0]
28. Hanne J, Liu J, Lee JB, Fishel R. Single-molecule FRET studies on DNA mismatch repair. Int J Biophys. 2013;3(1A):18-38. [Link]
29. Kulinski J, Besack D, Oleykowski CA, Godwin AK, Yeung AT. CEL I enzymatic mutation detection assay. Biotechniques. 2000;29(1):44-6, 48. [Link] [DOI:10.2144/00291bm07]

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.