Iranian
Biomedical Journal
Volume 26, Issue 1 (1-2022)
IBJ 2022, 26(1): 85-90 |
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Hezarjaribi N, Fazeli M R. A Simplified Process for Purification and Refolding of Recombinant Human Interferon-α2b. IBJ 2022; 26 (1) :85-90
URL: http://ibj.pasteur.ac.ir/article-1-3476-en.html
URL: http://ibj.pasteur.ac.ir/article-1-3476-en.html
Abstract:
Background: Interferon α-2b is a vital biotherapeutic produced through the recombinant DNA technology in E. coli. The recombinant IFN-α2b normally appears as intercellular IBs, which requires intensive refolding and purification steps. Method: Purification of IFN-α2b from solubilized IB was performed using two-phase extraction. To optimize refolding conditions, the effects of pH and different additives, including cysteine, cystine, urea, glycerol, Triton X-100, NaCl, and arginine, were investigated. Optimal refolding buffer (0.64 mM of urea, 5.57 mM of cysteine , and 1.8 mM of cystine) was obtained using RSM. The refolding process was performed by an optimized refolding buffer in the dilution and fed-batch refolding method at different protein concentrations (25-1000 µg/mL). Result: At a final protein concentration of 500 µg/mL, the fed-batch refolding method yielded in a biological activity of 2.24 × 108 IU/mg, which was nearly twice that of dilution method. Conclusion: Fed-batch refolding method resulted in the biologically active IFN-α2b with high purity, which can be used for research and industrial purposes.
Type of Study: Full Length/Original Article |
Subject:
Pharmaceutical Biotechnology
References
1. Gull I, Aslam MS, Tipu I, Mushtaq R, Ali TZ, Athar MA. Development of latent Interferon alpha 2b as a safe therapeutic for treatment of hepatitis C virus infection. Scientific reports 2019; 9(1): 1-12. [DOI:10.1038/s41598-019-47074-y]
2. Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial cell factories 2015; 14(1): 1-10. [DOI:10.1186/s12934-015-0222-8]
3. Asmana Ningrum R. Human interferon alpha-2b: a therapeutic protein for cancer treatment. Scientifica 2014; 2014: 970315. [DOI:10.1155/2014/970315]
4. Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein expression and purification 2003; 28(1): 1-8. [DOI:10.1016/S1046-5928(02)00641-1]
5. Clark ED. Protein refolding for industrial processes. Current opinion in biotechnology 2001; 12(2): 202-207. [DOI:10.1016/S0958-1669(00)00200-7]
6. Patel, Ramesh N. Biocatalysis in the Pharmaceutical and Biotechnology Industries. Florida: CRC press; 2006. [DOI:10.1201/9781420019377]
7. Yamaguchi H, Miyazaki M. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies. Biomolecles 2014; 4(1): 235-251. [DOI:10.3390/biom4010235]
8. Yang Z, Zhang L, Zhang Y, Zhang T, Feng Y, Lu X, Lan W, Wang J, Wu H, Cao C, Wang X. Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method. PloS one 2011; 6(7): e22981. [DOI:10.1371/journal.pone.0022981]
9. Mannall GJ, Titchener-Hooker NJ, Dalby PA. Factors affecting protein refolding yields in a fed-batch
10. and batch-refolding system. Biotechnology and bioengineering. 2007; 97(6):1523-1534. [DOI:10.1002/bit.21377]
11. Fazeli A, Shojaosadati SA, Fazeli MR, Ilka H. Effect of parallel feeding of oxidizing agent and protein on fed-batch refolding process of recombinant interferon beta-1b. Process biochemistry 2011; 46(3): 796-800. [DOI:10.1016/j.procbio.2010.11.007]
12. Zhang T, Xu X, Shen L, Feng Y, Yang Z, Shen Y, Wang J, Jin W, Wang X. Modeling of protein refolding from inclusion bodies. Acta biochimica et biophysica sinica 2009; 41(12): 1044-1052. [DOI:10.1093/abbs/gmp098]
13. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-685. [DOI:10.1038/227680a0]
14. Greenfield NJ. Using circular dichroism spectra to estimate protein secondary structure. Nature protocols 2006; 1(6): 2876- 2890. [DOI:10.1038/nprot.2006.202]
15. Tiwari K, Shebannavar S, Kattavarapu K, Pokalwar S, Mishra MK, Chauhan UK. Refolding of recombinant human granulocyte colony stimulating factor: Effect of cysteine/cystine redox system. Indian journal of biochemistry and biophysics 2012; 49(4): 285-488.
16. Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. Journal of bioscience and bioengineering 2005; 99(4): 303-310. [DOI:10.1263/jbb.99.303]
17. Ho JG, Middelberg AP, Ramage P, Kocher HP. The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient. Protein science 2003; 12(4): 708-716. [DOI:10.1110/ps.0233703]
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