[Home ] [Archive]   [ فارسی ]  
:: Main :: About :: Current Issue :: Archive :: Search :: Submit :: Contact ::
:: Volume 29, Issue 5 (11-2021) ::
Journal of Ilam University of Medical Sciences 2021, 29(5): 63-74 Back to browse issues page
Optimization of the Refolding Process for Recombinant Anti-EGFR Immunotoxin Produced in the Escherichia coli
Bahman Akbari *
Dept of Medical Biotechnology, Faculty of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran , ba1389@yahoo.com
Abstract:   (656 Views)
Introduction: Overexpression of the EGFR is associated with carcinogenesis, and it is observed in more than 70% of head and neck cancers. The expression of an immunotoxin against EGFR designed as an alternative to full antibody led to the production of aggregated protein in the form of inclusion bodies. This study aimed to investigate the 8M urea and 6M guanidine hydrochloride approaches for obtaining the immunotoxin as the soluble and effective form with correct folding.
Material & Methods: The BL21 (DE3) cells containing the pET28a-huimmunotoxin construct were induced by 1 mM IPTG at 37°C for 24 h, and the amount of expression was checked by SDS-PAGE. This immunotoxin was in the form of inclusion bodies and was solubilized individually in 8 M urea and 6 M guanidine hydrochloride and then purified by Ni-NTA affinity chromatography, which was observed as a single band in SDS-PAGE analysis. To correctly refold the obtained immunotoxin, the purified samples were poured into a dialysis bag, and denaturing agents were removed in a multi-step process called stepwise dialysis. The reactivity assessment of the purified and refold immunotoxin was assessed by ELISA technique using A431 cell lysate.
Findings: The immunotoxin (17 mg/ml) was expressed using the bacteria cells in the form of inclusion bodies. The refolded humanized immunotoxin had a high reactivity with A431 cells, indicating the suitable folding of the purified immunotoxin. The 50% binding activity rates of humanized immunotoxin obtained from urea and guanidine hydrochloride approaches were 0.8 and 1.7 µg/ml, respectively.
Discussion & Conclusion: The results of this study revealed that the urea approach was very effective in solubilizing and proper refolding of immunotoxins that were expressed in bacteria cells as inclusion bodies
Keywords: Guanidine hydrochloride, Inclusion body, Refolding, Single chain antibody, Urea
Full-Text [PDF 622 kb]   (221 Downloads)    
Type of Study: Research | Subject: biotechnolohgy
Received: 2021/06/1 | Accepted: 2021/09/21 | Published: 2021/11/8
References
1. Mathew M, Verma RS. Humanized immunotoxins a new generation of immunotoxins for targeted cancer therapy. Cancer Sci2009;100:1359-65. doi. 10.1111/j.1349-7006.2009.01192.x
2. Uribe ML, Marrocco I, Yarden Y. EGFR in cancer signaling mechanisms drugs and acquired resistance. Cancers2021;13:2748. doi.10.3390/cancers13112748
3. Li YM, Hall WA. Targeted toxins in brain tumor therapy. Toxins2010;2:2645-62. doi.10.3390/toxins2112645
4. Davies RL, Grosse VA, Kucherlapati R, Bothwell M. Genetic analysis of epidermal growth factor action: assignment of human epidermal growth factor receptor gene to chromosome 7. Proc NatI Acad Sci1980;77:4188-92. doi. 10.1073/pnas.77.7.4188
5. Mckenna N. Challenges in scale up of antibody production companies overcoming obstacles In production process. Genet Eng2001;21:10.
6. Li M, Su ZG, Janson JC. Invitro protein refolding by chromatographic procedures. Prot Expr Pur2004;33:1-10. doi.10.1016/j.pep.2003.08.023
7. Tsumoto K, Shinoki K, Kondo H, Uchikawa M, Juji T, Kumagai I. Highly efficient recovery of functional single chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent application to a human single chain Fv fragment. J Immunol Meth1998;219:119-29. doi.10.1016/s0022-1759(98)00127-6
8. Sinacola JR, Robinson AS. Rapid refolding and polishing of single chain antibodies from Escherichia coli inclusion bodies. Prot Expr Pur2002;26:301-8. doi.10.1016/s1046-5928(02)00538-7
9. Menezes MA, Aires KA, Ozaki CY, Ruiz RM, Pereira MC, Abreu PA, et al. Cloning approach and functional analysis of anti intimin single chain variable fragment. BMC Res Notes2011;4:1. doi.10.1186/1756-0500- -30
10. Zettlmeissl G, Rudolph R, Jaenicke R. Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 1. Physical properties and kinetics of aggregation. Biochemistry1979;18 :5567-71. doi.10.1021/bi00592a007
11. Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng2005;99:303-10. doi. 10.1263/jbb.99.303
12. Gerami SM, Farajnia S, Mahboudi F, Babaei H. Optimizing refolding condition for recombinant tissue plasminogen activator. Iran J Biotechnol 2011;9:253-9.
13. Goodman M. Market watch sales of biologics to show robust growth through to 2013. Nat Rev Drug Discov2009; 8:837. doi.10.1038/nrd3040
14. Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv2009;27:297-306. doi. 10.1016/j.biotechadv.2009.01.008
15. Bhopale G, Nanda R. Recombinant DNA expression products for human therapeutic use. Curr Sci2005;89:614-22.
16. Zhang L, Chou CP, Mooyoung M. Disulfide bond formation and its impact on the biological activity and stability of recombinant therapeutic proteins produced by Escherichia coli expression system. Biotechnol Adv2011;29:923-9. doi.10.1016/j.biotechadv.2011.07.013
17. Walsh G. Biopharmaceuticals: recent approvals and likely directions. Trend Biotechnol2005;23:553-8. doi.10.1016/j.tibtech.2005.07.005
18. Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Prot Exp Pur2003;28:1-8. doi.10.1016/s1046-5928(02)00641-1
19. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol2012;2012. doi.10.1155/2012/980250
20. Weisser NE, Hall JC. Applications of single chain variable fragment antibodies in therapeutics and diagnostics. Biotechnol Adv2009;27:502-20. doi.10.1016/j.biotechadv.2009.04.004
21. Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol2004;22:1399-408. doi.10.1038/nbt1029.
22. Villaverde A, Carrio MM. Protein aggregation in recombinant bacteria biological role of inclusion bodies. Biotechnol let2003;25:1385-95. doi.10.1023/a:1025024104862
23. Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, et al. Highly efficient production of soluble proteins from insoluble inclusion bodies by a two step denaturing and refolding method. PLos One2011;6:22981. doi.10.1371/journal.pone.0022981
24. Oganesyan N, Kim SH, Kim R. On column chemical refolding of proteins. Pharmacogenomics 2004;4:22-5.
25. Goldstein NI, Giorgio NA, Jones ST, Saldanha JW. Humanized anti EGF receptor monoclonal antibody. Patents; 20062:231-6.
26. Sun H, Wu G, Chen Y, Tian Y, Yue Y, Zhang G. Expression production and renaturation of a functional single chain variable antibody fragment against human ICAM1. Braz J. Med Biol Res2014;47:540-7. doi.10.1590/1414-431x20143276
27. Akbari B, Farajnia S, Zarghami N, Mahdieh N, Rahmati M, Khosroshahi SA, et al. Construction expression and activity of a novel immunotoxin comprising a humanized antiepidermal growth factor receptor scFv and modified Pseudomonas aeruginosa exotoxin A. Antican Drug2017;28:263-70. doi. 10.1097/CAD.0000000000000452
28. Akbari B, Farajnia S, Zarghami N, Mahdieh N, Rahmati M, Khosroshahi SA, et al. Design expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor. Prot Exp Pur2016;127:8-15. doi.10.1016/j.pep.2016.06.001
29. Buchner J, Rudolph R. Renaturation, purification and characterization of recombinant F ab-fragments produced in Escherichia coli. Biotechnology 1991;9(2):157-62. doi.10.1038/nbt0291-157
30. Kim SH. Expression and purification of recombinant immunotoxin a fusion protein stabilizes a single chain Fv in denaturing condition. Prot Exp Pur2003;27:85-9. doi. 10.1016/s1046-5928(02)00539
Send email to the article author

Add your comments about this article
Your username or Email:

CAPTCHA

Ethics code: IR.KUMS.REC.1398.568



XML   Persian Abstract   Print


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

Akbari B. Optimization of the Refolding Process for Recombinant Anti-EGFR Immunotoxin Produced in the Escherichia coli. Journal of Ilam University of Medical Sciences 2021; 29 (5) :63-74
URL: http://sjimu.medilam.ac.ir/article-1-7119-en.html


Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Volume 29, Issue 5 (11-2021) Back to browse issues page
مجله دانشگاه علوم پزشکی ایلام Journal of Ilam University of Medical Sciences
Persian site map - English site map - Created in 0.14 seconds with 31 queries by YEKTAWEB 4541