Reports of Biochemistry
and Molecular Biology
Mon, Dec 11, 2023
[Archive]
Volume 12, Issue 1 (Vol.12 No.1 Apr 2023)
rbmb.net 2023, 12(1): 27-35 |
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:
Hashemzaei M, Negahdaripour M, Heidari R, Ghoshoon* M B. Protein Expression and Purification of Romiplostim and Analysis of Its Secretory Production Using an In Silico Investigated Signal Peptide in E. Coli. rbmb.net 2023; 12 (1) :27-35
URL: http://rbmb.net/article-1-1086-en.html
URL: http://rbmb.net/article-1-1086-en.html
Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran & Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
Abstract: (972 Views)
Background: Romiplostim is a thrombopoietin receptor agonist approved for the treatment of immune thrombocytopenia. It is produced by recombinant DNA technology in Escherichia coli. Many researchers have studied the periplasmic or extracellular production of recombinant proteins in E. coli by using signal peptide sequences due to its advantages compared to intracellular production. In this study, the effect of the pelB signal peptide on Romiplostim production was analyzed.
Methods: The nucleotide sequence of Romiplostim was codon optimized for expression in E. coli BL21. For analysis of the effect of the pelB signal peptide, pET-22b (+) and pET-15b plasmids were used. The probability of signal peptide cleavage and pathway was predicted by using the SignalP 5.0 program, and expression, purification, and biological activity of the recombinant protein were analyzed.
Results: In-silico analysis predicted the correct cleavage of the pelB signal peptide. However, the experimental results showed intracellular accumulation of the protein in fusion with this signal peptide without any detectable protein band in periplasmic or extracellular spaces. The in-vivo experiment of purified protein without signal peptide exhibited a significant increment in platelets compared to the control group.
Conclusions: Romiplostim was expressed in E. coli with and without signal peptide. The latest one showed suitable in-vivo bioactivity. Despite the results of in-silico prediction, the pelB signal peptide could not transport the protein into the periplasm or extracellular environment in the experimental condition. Trying different signal peptides and more in-silico analysis might be helpful for the efficient secretion of the Romiplostim protein.
Methods: The nucleotide sequence of Romiplostim was codon optimized for expression in E. coli BL21. For analysis of the effect of the pelB signal peptide, pET-22b (+) and pET-15b plasmids were used. The probability of signal peptide cleavage and pathway was predicted by using the SignalP 5.0 program, and expression, purification, and biological activity of the recombinant protein were analyzed.
Results: In-silico analysis predicted the correct cleavage of the pelB signal peptide. However, the experimental results showed intracellular accumulation of the protein in fusion with this signal peptide without any detectable protein band in periplasmic or extracellular spaces. The in-vivo experiment of purified protein without signal peptide exhibited a significant increment in platelets compared to the control group.
Conclusions: Romiplostim was expressed in E. coli with and without signal peptide. The latest one showed suitable in-vivo bioactivity. Despite the results of in-silico prediction, the pelB signal peptide could not transport the protein into the periplasm or extracellular environment in the experimental condition. Trying different signal peptides and more in-silico analysis might be helpful for the efficient secretion of the Romiplostim protein.
Type of Article: Original Article |
Subject:
Molecular Biology
Received: 2022/10/30 | Accepted: 2023/01/22 | Published: 2023/08/15
Received: 2022/10/30 | Accepted: 2023/01/22 | Published: 2023/08/15
References
1. Cines DB, Yasothan U, Kirkpatrick P. Romiplostim. Nat Rev Drug Discov. 2008;7(11). [DOI:10.1038/nrd2741] [PMID]
2. Cines DB, Blanchette VS. Immune thrombocytopenic purpura. N Engl J Med. 2002;346(13):995-1008. [DOI:10.1056/NEJMra010501] [PMID]
3. Psaila B, Bussel JB. Immune thrombocytopenic purpura. Hematol Oncol Clin North Am. 2007;21(4):743-59. [DOI:10.1016/j.hoc.2007.06.007] [PMID]
4. Stasi R, Provan D. Management of immune thrombocytopenic purpura in adults. Mayo Clin Proc. 2004;79(4):504-22. [DOI:10.4065/79.4.504] [PMID]
5. McMillan R. The pathogenesis of chronic immune thrombocytopenic purpura. Semin Hematol. 2007;44(4 Suppl 5):S3-S11. [DOI:10.1053/j.seminhematol.2007.11.002] [PMID]
6. Zainal A, Salama A, Alweis R. Immune thrombocytopenic purpura. J Community Hosp Intern Med Perspect. 2019;9(1):59-61. [DOI:10.1080/20009666.2019.1565884] [PMID] [PMCID]
7. Calmettes C, Vigouroux S, Tabrizi R, Milpied N. Romiplostim (AMG531, Nplate) for secondary failure of platelet recovery after allo-SCT. Bone Marrow Transplant. 2011;46(12):1587-9. [DOI:10.1038/bmt.2011.179] [PMID]
8. Nplate (romiplostim) information [Internet]. U.S. Food and Drug Administration. FDA; [cited 2023 Mar 1]. Available from: https://www.fda.gov/drugs/postmarket-drug-safety-information-patients-and-providers/nplate-romiplostim-information.
9. Janssens A. Romiplostim for the treatment of primary immune thrombocytopenia. Expert Rev Hematol. 2012;5(2):133-44. [DOI:10.1586/ehm.12.6] [PMID]
10. Choi J, Lee S. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol. 2004;64:625-35. [DOI:10.1007/s00253-004-1559-9] [PMID]
11. Ghoshoon MB, Berenjian A, Hemmati S, Dabbagh F, Karimi Z, Negahdaripour M, et al. Extracellular production of recombinant L-Asparaginase II in Escherichia coli: Medium optimization using response surface methodology. Int J Pept Res Ther. 2015;21:487-95. [DOI:10.1007/s10989-015-9476-6]
12. Kleiner‐Grote GR, Risse JM, Friehs K. Secretion of recombinant proteins from E. coli. Eng Life Sci. 2018;18(8):532-50. [DOI:10.1002/elsc.201700200] [PMID] [PMCID]
13. Yoon SH, Kim SK, Kim JF. Secretory production of recombinant proteins in Escherichia coli. Recent Pat Biotechnol. 2010;4(1):23-9. [DOI:10.2174/187220810790069550] [PMID]
14. Pechsrichuang P, Songsiriritthigul C, Haltrich D, Roytrakul S, Namvijtr P, Bonaparte N, et al. OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system. Springerplus. 2016;5(1):1-10. doi: 10.1186/s40064-016-2893-y. [DOI:10.1186/s40064-016-2893-y] [PMID] [PMCID]
15. Mirzadeh K, Shilling PJ, Elfageih R, Cumming AJ, Cui HL, Rennig M, et al. Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microb Cell Fact. 2020;19:1-12. [DOI:10.1186/s12934-020-01339-8] [PMID] [PMCID]
16. Owji H, Nezafat N, Negahdaripour M, Hajiebrahimi A, Ghasemi Y. A comprehensive review of signal peptides: Structure, roles, and applications. Eur J Cell Biol. 2018;97(6):422-41. [DOI:10.1016/j.ejcb.2018.06.003] [PMID]
17. Wang J, Xu S, Pang Y, Wang X, Chen K, Ouyang P. Highly Efficient Extracellular Production of Recombinant Streptomyces PMF Phospholipase D in Escherichia coli. Catalysts. 2020;10(9):1057. [DOI:10.3390/catal10091057]
18. Zamani M, Nezafat N, Negahdaripour M, Dabbagh F, Ghasemi Y. In silico evaluation of different signal peptides for the secretory production of human growth hormone in E. coli. Int J Pept Res Ther. 2015;21:261-8. [DOI:10.1007/s10989-015-9454-z]
19. Chuan-Fa Liu, Ulrich Feige, Cheetham JC, inventors. Thrombopoietic compounds. USA patent US6835809B1. 2004-12-28.
20. Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. 2007;(6):253. doi: 10.3791/253. [DOI:10.3791/253] [PMID] [PMCID]
21. Ghasemi Y, Ghoshoon MB, Taheri M, Negahdaripour M, Nouri F. Cloning, expression and purification of human PDGF-BB gene in Escherichia coli: New approach in PDGF-BB protein production. Gene Rep. 2020;19:100653. [DOI:10.1016/j.genrep.2020.100653]
22. Negahdaripour M, Nezafat N, Heidari R, Erfani N, Hajighahramani N, Ghoshoon MB, et al. Production and preliminary in vivo evaluations of a novel in silico-designed L2-based potential HPV vaccine. Curr Pharm Biotechnol. 2020;21(4):316-24. [DOI:10.2174/1389201020666191114104850] [PMID]
23. Gallagher SR. SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE). Curr Protoc Essent Lab Tech. 2012;6(1):7-3. [DOI:10.1002/9780470089941.et0703s06]
24. Hober S, Nord K, Linhult M. Protein A chromatography for antibody purification. J Chromatogr B. 2007;848(1):40-7. [DOI:10.1016/j.jchromb.2006.09.030] [PMID]
25. Fayaz S, Fard-Esfahani P, Golkar M, Allahyari M, Sadeghi S. Expression, purification and biological activity assessment of romiplostim biosimilar peptibody. Daru. 2016;24(1):1-5. [DOI:10.1186/s40199-016-0156-7] [PMID] [PMCID]
26. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. [DOI:10.1016/0003-2697(76)90527-3] [PMID]
27. Sadraeian M, Rasoul-Amini S, Mansoorkhani MJK, Mohkam M, Ghoshoon MB, Ghasemi Y. Induction of antitumor immunity against cervical cancer by protein HPV-16 E7 in fusion with ricin B chain in tumor-bearing mice. Int J Gynecol Cancer. 2013;23(5):809-14. [DOI:10.1097/IGC.0b013e3182907989] [PMID]
28. Arakawa T, Ejima D. Refolding technologies for antibody fragments. Antibodies. 2014;3(2):232-41. [DOI:10.3390/antib3020232]
29. Kötzler MP, McIntosh LP, Withers SG. Refolding the unfoldable: A systematic approach for renaturation of Bacillus circulans xylanase. Protein Sci. 2017;26(8):1555-63. [DOI:10.1002/pro.3181] [PMID] [PMCID]
30. Li A, Kato Z, Ohnishi H, Hashimoto K, Matsukuma E, Omoya K, et al. Optimized gene synthesis and high expression of human interleukin-18. Protein Expr Purif. 2003;32(1):110-8. [DOI:10.1016/j.pep.2003.08.003] [PMID]
31. Karimi Z, Nezafat N, Negahdaripour M, Berenjian A, Hemmati S, Ghasemi Y. The effect of rare codons following the ATG start codon on expression of human granulocyte-colony stimulating factor in Escherichia coli. Protein Expr Purif. 2015;114:108-14. [DOI:10.1016/j.pep.2015.05.017] [PMID]
32. Chung BH, Sohn M-J, Oh S-W, Park U-S, Poo H, Kim BS, et al. Overproduction of human granulocyte-colony stimulating factor fused to the PelB signal peptide in Escherichia coli. J Ferment Bioeng. 1998;85(4):443-6. [DOI:10.1016/S0922-338X(98)80092-5]
33. Su L, Xu C, Wu J. Extracellular expression of Thermobifida fusca cutinase with pelB signal peptide depends on more than type II secretion pathway in Escherichia coli. J Biotechnol. 2015;204:47-52. [DOI:10.1016/j.jbiotec.2015.03.029] [PMID]
34. Shi L, Liu H, Gao S, Weng Y, Zhu L. Enhanced extracellular production of is PETase in Escherichia coli via engineering of the pelB signal peptide. J Agric Food Chem. 2021;69(7):2245-52. [DOI:10.1021/acs.jafc.0c07469] [PMID]
35. Negahdaripour M, Nezafat N, Hajighahramani N, Soheil Rahmatabadi S, Hossein Morowvat M, Ghasemi Y. In silico study of different signal peptides for secretory production of interleukin-11 in Escherichia coli. Curr Proteomics. 2017;14(2):112-21. [DOI:10.2174/1570164614666170106110848]
36. Freudl R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact. 2018;17(1):52. [DOI:10.1186/s12934-018-0901-3] [PMID] [PMCID]
37. Hashemzaei M, Nezafat N, Ghoshoon MB, Negahdaripour M. In-silico selection of appropriate signal peptides for romiplostim secretory production in Escherichia coli. Inform Med Unlocked. 2023;36:101146. [DOI:10.1016/j.imu.2022.101146]
38. Mohammadi F, Nezafat N, Berenjian A, Negahdaripour M, Zamani M, Ghoshoon MB, et al. Extracellular production of a potent and chemically resistant nattokinase in immobilized Escherichia coli using response surface methodology. Curr Pharm Biotechnol. 2018;19(11):856-68. [DOI:10.2174/1389201019666181022115405] [PMID]
Send email to the article author
| Rights and permissions | |
 |
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
