Production of Recombinant E.coli Asparaginase on a Laboratory Scale

Document Type : Original Article(s)

Authors

1 Pharmacy Student, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Science, Isfahan, Iran

2 Assistant Professor, Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Background: L-asparaginase is an enzyme derived from E. coli and is one of the drugs used to treat acute lymphoblastic leukemia. Decreased blood asparagine after the use of the asparaginase can lead to death for these cells. The aim of this project is to investigate the possibility of the production and purification of theasparaginase enzyme.
Methods: The asparaginase gene of E. coli was amplified using PCR technique. After purification, the gene was cloned into pET28a vector and transformed into a bacterial host. After transformation, different protein expression conditions were investigated for optimization. Recombinant enzyme expression was confirmed by SDS-PAGE and Western blotting. The asparaginase enzyme was then purified by affinity chromatography using a column containing Ni-NTA resin and a hybrid method and finally, its enzymatic activity was investigated.
Findings: The high production of asparaginase enzyme in E. coli was at 37 °C, 1 mM concentration of IPTG, and incubation 20 hours after induction. Insoluble asparaginase enzyme was purified using a Ni-NTA column and the refolding process was performed on the column. According to the activity of the refolded enzyme in the enzyme activity test, it can be concluded that the refolding process of the enzyme has been done correctly.
Conclusion: Due to the great importance of asparaginase production in Iran and the need of a group of patients to use this enzyme, in this project, by optimizing the expression and purification process, the L-asparaginase enzyme was produced on a laboratory scale.

Keywords

References

  1. Brown PA, Shah B, Fathi A, Wieduwilt M, Advani A, Aoun P, et al. NCCN guidelines insights: acute lymphoblastic leukemia, version 1.2017. J Natl Compr Canc Netw 2017; 15(9): 1091-102.
  2. De Kouchkovsky I, Abdul-Hay M. Acute myeloid leukemia: a comprehensive review and 2016 update. Blood Cancer J 2016; 6(7): e441.
  3. Mehta P. Adult acute lymphocytic leukemia: biology and treatment. JAMA 1978; 305(22): 2353-8.
  4. Rafei H, Kantarjian HM, Jabbour EJ. Recent advances in the treatment of acute lymphoblastic leukemia. Leuk Lymphoma 2019; 60(11): 2606-21.
  5. Pui CH, Relling MV, Downing JR. Acute lymphoblastic leukemia. N Engl J Med 2004; 350(15): 1535-48.
  6. Radadiya A, Zhu W, Coricello A, Alcaro S, Richards NGJ. Improving the treatment of acute lymphoblastic leukemia. Biochemistry. 2020; 59(35): 3193-200.
  7. Pieters R, Carroll WL. Biology and treatment of acute lymphoblastic leukemia. Pediatr Clin North Am 2008; 55(1): 1-20.
  8. Ebrahiminezhad A, Rasoul-Amini S, Ghasemi Y.
    l-Asparaginase production by moderate halophilic bacteria isolated from Maharloo Salt Lake. Indian J Microbiol 2011; 51(3): 307-11.
  9. Radha R, Arumugam N, Gummadi SN. Glutaminase free L-asparaginase from Vibrio cholerae: heterologous expression, purification and biochemical characterization. Int J Biol Macromol 2018; 111: 129-38.
  10. Cachumba JJM, Antunes FAF, Peres GFD, Brumano LP, Dos Santos JC, Da Silva SS. Current applications and different approaches for microbial
    L-asparaginase production. Braz J Microbiol 2016; 47(Suppl 1): 77-85.
  11. Vala AK, Sachaniya B, Dudhagara D, Panseriya HZ, Gosai H, Rawal R, et al. Characterization of
    L-asparaginase from marine-derived Aspergillus niger AKV-MKBU, its antiproliferative activity and bench scale production using industrial waste. Int J Biol Macromol 2018; 108: 41-6.
  12. Nunes JC, Cristóvão RO, Freire MG, Santos-Ebinuma VC, Faria JL, Silva CG, Tavares AP. Recent strategies and applications for L-asparaginase confinement. Molecules 2020; 25(24): 5827.
  13. Chand S, Mahajan RV, Prasad JP, Sahoo DK, Mihooliya KN, Dhar MS, et al. A comprehensive review on microbial l‐asparaginase: Bioprocessing, characterization, and industrial applications. Biotechnol Appl Biochem 2020; 67(4): 619-47.
  14. Alrumman SA, Mostafa YS, Al-Izran KA, Alfaifi MY, Taha TH, Elbehairi SE. Production and anticancer activity of an L-asparaginase from Bacillus licheniformis isolated from the Red Sea, Saudi Arabia. Sci Rep 2019; 9(1): 3756.
  15. Mikucki J, Sobiś M, Różalska M. Localisation of staphylococcal L-asparaginase. FEMS microbiology letters 1986; 37(2): 129-33.
  16. Abdel-Fattah YR, Olama ZA. L-asparaginase production by Pseudomonas aeruginosa in solid-state culture: evaluation and optimization of culture conditions using factorial designs. Process Biochemistry 2002; 38(1): 115-22.
  17. El-Bessoumy AA, Sarhan M, Mansour J. Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem Mol Biol 2004; 37(4):
    387-93.
  18. Sandhya P, Nikku MY, Chaduvula AIR, Dasari VRRK, Donthireddy SR. Application of statistical experimental designs for the optimization of medium constituents for the production of L-Asparaginase by serratia marcescens. J Microbial Biochem Technol 2010; 2(4).
Journal of Isfahan Medical School
Volume 40, Issue 696
4th Week,, January
January and February 2023
Pages 950-956

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History

  • Receive Date: 29 April 1401
  • Accept Date: 06 October 1401

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