Гетерологичная экспрессия генов рекомбинантных L-аспарагиназ

##plugins.themes.bootstrap3.article.sidebar##

pdf
Опубликован: Dec 5, 2025
DOI: https://doi.org/10.18097/BMCRM00265
Ключевые слова:
L-аспарагиназа; оптимизация экспрессии; гетерологичная экспрессия; рациональный и компьютерный дизайн; рекомбинантные гены

##plugins.themes.bootstrap3.article.main##

М.В. Покровская
Научно-исследовательский институт биомедицинской химии им. В.Н. Ореховича, 119121, Москва, ул. Погодинская, 10
С.С. Александрова
Научно-исследовательский институт биомедицинской химии им. В.Н. Ореховича, 119121, Москва, ул. Погодинская, 10
В.С. Покровский
Научно-исследовательский институт биомедицинской химии им. В.Н. Ореховича, 119121, Москва, ул. Погодинская, 10
Н.Д. Добрякова
Научно-исследовательский институт биомедицинской химии им. В.Н. Ореховича, 119121, Москва, ул. Погодинская, 10
А.Н. Шишпаренок
Научно-исследовательский институт биомедицинской химии им. В.Н. Ореховича, 119121, Москва, ул. Погодинская, 10
Ю.В. Гладилина
Научно-исследовательский институт биомедицинской химии им. В.Н. Ореховича, 119121, Москва, ул. Погодинская, 10
Д.Д. Жданов
Научно-исследовательский институт биомедицинской химии им. В.Н. Ореховича, 119121, Москва, ул. Погодинская, 10

Аннотация

L-аспарагиназa (КФ 3.5.1.1.) — фермент с самым высоким уровнем мирового производства, используемый при лечении онкологических заболеваний, а также в пищевой промышленности. Для производства многих целевых белков используются различные системы экспрессии – от бесклеточных до гиперпродуктивных клеток растений, насекомых, бактерий и млекопитающих. В данном обзоре предпринята попытка обобщить общирные данные по экспрессии гетерологичных генов и технологии производства рекомбинантных L-аспарагиназ.

##plugins.themes.bootstrap3.article.details##

Как цитировать
Покровская M., Александрова S., Покровский V., Добрякова N., Шишпаренок A., Гладилина Y., & Жданов D. (2025). Гетерологичная экспрессия генов рекомбинантных L-аспарагиназ. Biomedical Chemistry: Research and Methods, 8(4), e00265. https://doi.org/10.18097/BMCRM00265
Выпуск
Том 8 № 4 (2025): Принято в печать
Раздел
ОБЗОРЫ

Библиографические ссылки

  1. Loch, J., Jaskolski, M. (2021) Structural and biophysical aspects of L-asparaginases: A growing family with amazing diversity. IUCrJ, 8 (4), 514-531. DOI
  2. Brumano, L., da Silva, F.V.S., Costa-Silva, T., Apolinário, A., Santos, J., Kleingesinds, E., Monteiro, G., Rangel-Yagui, C., Benyahia, B., Junior, A. (2019) Development of L-asparaginase biobetters: current research status and review of the desirable quality profiles. Frontiers in bioengineering and biotechnology, 10(6), 212. DOI
  3. Cachumba, J.J., Antunes, F.A., Peres, G.F., Brumano, L.P., Santos, J.C., Da Silva, S.S. (2016) Current applications and different approaches for microbial L-asparaginase production. Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology], 47 (Suppl 1), 77-85. DOI
  4. Eisele, N., Linke, D., Bitzer, K., Na’amnieh, S., Nimtz, M., Berger, R. (2011) The first characterized asparaginase from a basidiomycete, Flammulina velutipes. Bioresource technology, 102(3), 3316-3321. DOI
  5. Jha, S. K., Pasrija, D., Sinha, R., Singh, H.R., Nigam, V., Vidyarthi, A. (2012) Microbial L-asparaginase: a review on current scenario and future prospects. International Journal of Pharmaceutical Sciences and Research, 3(9), 3076- 3090. DOI
  6. Dumina, M., Zhgun, A., Pokrovskaya, M., Aleksandrova, S., Zhdanov, D., Sokolov, N., El’darov, M. (2021) Highly active thermophilic L-asparaginase from Melioribacter roseus represents a novel large group of type II bacterial L-asparaginases from chlorobi-ignavibacteriae-bacteroidetes clade. International journal of molecular sciences, 22(24), 13632. DOI
  7. Mahajan, R.V., Kumar, V., Rajendran, V., Saran, S., Ghosh, P.C., Saxena, R.K. (2014) Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties. PLoS One, 9(6):e99037. DOI
  8. Sarquis, M.I., Oliveira, E.M., Santos, A.S., Costa, G.L. (2004) Production of L-asparaginase by filamentous fungi. Memorias do Instituto Oswaldo Cruz. 99(5), 489-492. DOI
  9. da Cunha, M.C, Dos Santos, Aguilar, J.G., de Melo, R.R., Nagamatsu, S.T., Ali, F., de Castro, R.J.S., Sato, H.H. (2019) Fungal L-asparaginase: Strategies for production and food applications. Food research international, 126, 108658. DOI
  10. Saleh, A.A., El-Aref, H.M., Ezzeldin, A.M., Ewida R.M., Bedak, O.A.Al. (2025) L-asparaginase from the novel Fusarium falciforme AUMC 16563: extraction, purification, characterization, and cytotoxic effects on PC-3, HePG- 2, HCT-116, and MCF-7 cell lines. BMC microbiology, 25(1), 145. DOI
  11. Casado, A., Caballero, J.L., Franco, A.R., Cárdenas, J., Grant, M.R., Muñoz-Blanco, J. (1995) Molecular cloning of the gene encoding the L-asparaginase gene of Arabidopsis thaliana. Plant physiology, 108(3), 1321- 1322. DOI
  12. Sharma, A., Kaushik., V., Goel, M. (2022) Insights into the distribution and functional properties of L-asparaginase in the Archaeal domain and characterization of Picrophilus torridus asparaginase belonging to the novel family Asp2like1. ACS Omega, 7(45), 40750-40765. DOI
  13. Broome, J.D. (1965) Antilymphoma activity of L-asparaginase in vivo: clearance rates of enzyme preparations from guinea pig serum and yeast in relation to their effect on tumor growth. Journal of the National Cancer Institute. 35(6), 967-974. DOI
  14. Lopes, A.M., Oliveira-Nascimento, L., Ribeiro, A., Tairum, C.A. Jr., Breyer, C.A., Oliveira, M.A., Monteiro, G., Souza-Motta, C.M., Magalhães, P.O., Avendaño, J.G., Cavaco-Paulo, A.M., Mazzola, P.G., Rangel-Yagui, C.O., Sette, L.D., Converti, A., Pessoa, A. (2017) Therapeutic L-asparaginase: upstream, downstream and beyond. Critical reviews in biotechnology, 37(1), 82-99. DOI
  15. Bosmann, H.B., Kessel, D. (1970) Inhibition of glycoprotein synthesis in L5178Y mouse leukaemic cells by L-asparaginase in vitro. Nature. 226(5248), 850-851. DOI
  16. Bejger, M., Imiolczyk, B., Clavel, D., Gilski, M., Pajak, A., Marsolais, F., Jaskolski, M. (2014) Na⁺/K⁺ exchange switches the catalytic apparatus of potassium-dependent plant L-asparaginase. Acta crystallographica. Section D, Biological crystallography, 70(Pt 7),1854-1872. DOI
  17. Vimal, A., Kumar, A. (2020) Antimicrobial potency evaluation of free and immobilized L-asparaginase using chitosan nanoparticles. Journal of Drug Delivery Science and Technology. 61(6), 102231. DOI
  18. Vimal, A., Kumar, A. (2022) L-asparaginase: Need for an expedition from an enzymatic molecule to antimicrobial drug. International journal of peptide research and therapeutics. 28(1), 9. DOI
  19. Zielezinski, A., Loch, J.I., Karlowski, W.M., Jaskolski, M. (2022) Massive annotation of bacterial L-asparaginases reveals their puzzling distribution and frequent gene transfer events. Scientific reports.12(1),15797. DOI
  20. Abd El-Baky, H.H., El-Baroty, G.S. (2020) Spirulina maxima L-asparaginase: immobilization, antiviral and antiproliferation activities. Recent patents on biotechnology, 14(2), 154-163. DOI
  21. Vimal, A., Kumar, A. (2018) L-Asparaginase: a feasible therapeutic molecule for multiple diseases. 3 Biotech, 8(6), 278. DOI
  22. Darvishi, F., Jahanafrooz, Z., Mokhtarzadeh, A. (2022) Microbial L-asparaginase as a promising enzyme for treatment of various cancers. Applied microbiology and biotechnology, 106(17), 5335-5347. DOI
  23. Ściuk, A., Wątor, K., Staroń, I., Worsztynowicz, P., Pokrywka, K., Sliwiak, J., Kilichowska, M., Pietruszewska, K., Mazurek, Z., Skalniak, A., Lewandowski, K., Jaskolski, M., Loch, J.I., Surmiak, M. (2024). Substrate affinity is not crucial for therapeutic L-asparaginases: antileukemic activity of novel bacterial enzymes. Molecules (Basel, Switzerland), 29(10), 2272. DOI
  24. Wang, N., Ji, W., Wang, L., Wu, W., Zhang, W., Wu, Q., Du, W., Bai, H., Peng, B., Ma, B., Li, L. (2022) Overview of the structure, side effects, and activity assays of L-asparaginase as a therapy drug of acute lymphoblastic leukemia. RSC medicinal chemistry, 13(2), 117-128. DOI
  25. Patel, P., Panseriya, H., Vala, A.K., Dave, B.P., Gosai, H. (2022). Exploring current scenario and developments in the field of microbial L-asparaginase production and applications: A review. Process Biochemistry, 121, 529-541. DOI
  26. Xu, F., Oruna-Concha, M.J., Elmore, J.S. (2016) The use of asparaginase to reduce acrylamide levels in cooked food. Food chemistry. 210, 163-171. DOI
  27. Santos, J.H.P.M., Costa, I.M., Molino, J.V.D., Leite, M.S.M., Pimenta, M.V., Coutinho, J.A.P., Pessoa, A.Jr., Ventura, S.P.M., Lopes, A.M., Monteiro, G. (2017) Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in E. coli host. Biotechnology progress, 33(2), 416- 424. DOI
  28. Tekoah, Y., Shulman, A., Kizhner, T., Ruderfer, I., Fux, L., Nataf, Y., Bartfeld, D., Ariel, T., Gingis-Velitski, S., Hanania, U., Shaaltiel, Y. (2015) Largescale production of pharmaceutical proteins in plant cell culture-the Protalix experience. Plant biotechnology journal. 13(8), 1199-1208. DOI
  29. Zhu, J. (2012) Mammalian cell protein expression for biopharmaceutical production. Biotechnology advances, 30(5), 1158-1170. DOI
  30. Zhang, X. Wang, Z., Wang, Y., Li, X., Zhu, M., Zhang, H., Xu, M., Yang, T., Rao, Z. (2021) Heterologous expression and rational design of L-asparaginase from Rhizomucor miehei to improve thermostability. Biology, 10(12), 1346. DOI
  31. Lefin, N., Miranda, J., Beltrán, J.F., Belén, L.H., Effer, B., Pessoa, A. Jr., Farias, J.G., Zamorano, M. (2023) Current state of molecular and metabolic strategies for the improvement of L-asparaginase expression in heterologous systems. Frontiers in pharmacology, 14, 1208277. DOI
  32. Yang, X., Rao, Y., Zhang, M., Wang, J., Liu, W., Cai, D., Chen, S. (2023) Efficient production of L-asparaginase in Bacillus licheniformis by optimizing expression elements and host. Chinese journal of biotechnology, 39(3), 1096- 1106. DOI
  33. Li, X., Xu, S., Zhang, X., Xu, M., Yang, T., Wang, L., Zhang, H., Fang, H., Osire, T., Yang, S., Rao, Z. ( 2019) Design of a high-efficiency synthetic system for L-asparaginase production in Bacillus subtilis. Engineering in life sciences, 19(3), 229-239. DOI
  34. Costa-Silva, T.A., Camacho-Córdova, D.I., Agamez-Montalvo, G.S., Parizotto, L.A., Sánchez-Moguel, I., Pessoa-Jr, A. (2019) Optimization of culture conditions and bench-scale production of anticancer enzyme L-asparaginase by submerged fermentation from Aspergillus terreus CCT 7693. Preparative biochemistry & biotechnology, 49(1), 95-104. DOI
  35. Sharma, D., Mishra, A. (2023) Synergistic effects of ternary mixture formulation and process parameters optimization in a sequential approach for enhanced L-asparaginase production using agro-industrial wastes. Environmental science and pollution research international, 31(12), 1-16. DOI
  36. Poluri, K.M., Gulati, K. (2017) Rational designing of novel proteins through computational approaches. In: Protein engineering techniques.Springer Briefs in Applied Sciences and Technology. Springer Singapore. pp. 61-83. DOI
  37. Praveen, P. (2019). Modeling and validation of L-asparaginase enzyme, an anticancer agent using the tools of computational biology. International Journal of Research in Medical Sciences, 8(1), 211-214, DOI
  38. Kelley, L.A., Mezulis, S., Yates, C.M., Wass, M.N., Sternberg, M.J. (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nature protocols, 10(6), 845-858. DOI
  39. Gileadi, O. (2017) Recombinant protein expression in E. coli : A historical perspective. Methods in molecular biology, 1586, 3-10. DOI
  40. Saberianfar, R., Menassa, R. (2018) Strategies to increase expression and accumulation of recombinant proteins. In: Molecular Pharming: Applications, Challenges, and Emerging Areas. ( A.R. Kermode and L. Jiang eds.) New York. pp. 119-135. DOI
  41. Shishparenok, A.N., Gladilina, Y.A., Zhdanov, D.D. (2023) Engineering and expression strategies for optimization of L-asparaginase development and production. International journal of molecular sciences, 24(20),15220. DOI
  42. Miranda, J., Lefin, N., Beltran, J., Belén, L.H., Tsipa, A., Farias, J.G., Zamorano, M. (2023) Enzyme engineering strategies for the bioenhancement of L-asparaginase used as a biopharmaceutical. BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy, 37(6), 793-811. DOI
  43. Borek, D., Jaskólski, M. (2001) Sequence analysis of enzymes with asparaginase activity. Acta biochimica Polonica, 48(4), 893-902. DOI
  44. Michalska, K., Jaskolski, M. (2006). Structural aspects of L-asparaginases, their friends and relations. Acta biochimica Polonica, 53 (4), 627-640. DOI
  45. Castro, D., Marques, A., Almeida, M.R., de Paiva, G.B., Bento, H.B.S., Pedrolli, D.B., Freire, M.G., Tavares, A.P.M., Santos-Ebinuma, V.C. (2021) L-asparaginase production review: bioprocess design and biochemical characteristics. Applied microbiology and biotechnology, 105(11), 4515-4534. DOI
  46. Bonthron, D.T., Jaskólski, M. (1997) Why a “benign” mutation kills enzyme activity. Structure-based analysis of the A176V mutant of Saccharomyces cerevisiae L-asparaginase I. Acta biochimica Polonica, 44(3), 491-504. DOI
  47. Lubkowski, J., Wlodawer, A. (2021) Structural and biochemical properties of L-asparaginase. The FEBS journal, 288(14), 4183-4209. DOI
  48. da Silva, L.S., Doonan, L.B., Pessoa, A. Jr., de Oliveira, M.A., Long, P.F. (2022) Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature. Biotechnology and applied biochemistry, 69(2), 503-513. DOI
  49. Yun, M.K., Nourse, A., White, S.W., Rock, C.O., Heath, R.J. (2007) Crystal structure and allosteric regulation of the cytoplasmic E. coli L-asparaginase I. Journal of molecular biology, 369(3), 794-811. DOI
  50. Jennings, M.P., Beacham, I.R. (1993) Co-dependent positive regulation of the ansB promoter of E. coli by CRP and the FNR protein: a molecular analysis. Molecular microbiology, 9(1), 155-64. DOI
  51. Dunlop, P.C., Meyer, G.M., Ban, D., Roon, R.J. (1978) Characterization of two forms of asparaginase in Saccharomyces cerevisiae. The Journal of biological chemistry, 253(4), 1297-1304. DOI
  52. Dumina, M., Zhgun, A. (2023) Thermo-L-asparaginases: from the role in the viability of thermophiles and hyperthermophiles at high temperatures to a molecular understanding of their thermoactivity and thermostability. International journal of molecular sciences, 24(3), 2674. DOI
  53. Pokrovskaya, M.V., Pokrovsky, V.S., Aleksandrova, S.S., Sokolov, N.N., Zhdanov, D.D. (2022) Molecular analysis of L-asparaginases for clarification of the mechanism of action and optimization of pharmacological functions. Pharmaceutics, 14(3), 599. DOI
  54. Kotzia, G.A., Lappa, K., Labrou, N.E. ( 2007) Tailoring structure-function properties of L-asparaginase: engineering resistance to trypsin cleavage. The Biochemical journal, 404(2), 337-343. DOI
  55. Gesto, D.S., Cerqueira, N.M., Fernandes, P.A., Ramos, M.J. (2013) Unraveling the Enigmatic Mechanism of L-asparaginase II with Q M/QM Calculations. Journal of the American Chemical Society, 135(19), 7146-7158. DOI
  56. Aghaiypour, K., Wlodawer, A., Lubkowski, J. (2001) Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase. Biochemistry, 40(19), 5655-5664. DOI
  57. Upadhyay, A.K., Singh, A., Mukherjee, K.J., Panda, A.K. (2014) Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein. Frontiers in Microbiology, 5, 486. DOI
  58. Maurizi, M.R. (1992) Proteases and protein degradation in Escherichia coli. Experientia, 48(2), 178-201. DOI
  59. Wülfing, C., Plückthun, A. (1994) Protein folding in the periplasm of Escherichia coli. Molecular microbiology, 12(5), 685-692. DOI
  60. Papageorgiou, A.C., Posypanova, G.A., Andersson, C.S., Sokolov, N.N., Krasotkina, J. (2008) Structural and functional insights into Erwinia carotovora L-asparaginase. The FEBS journal, 275(17), 4306-4316. DOI
  61. Swain, A.L., Jaskólski, M., Housset, D., Rao, J.K., Wlodawer, A. (1993) Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proceedings of the National Academy of Sciences of the United States of America, 90(4), 1474-1478. DOI
  62. Pokrovskaya, M.V., Pokrovskiy, V.S., Aleksandrova, S.S, Anisimova, N.Iu., Andrianov, R.M., Treschalina, E.M., Ponomarev, G.V., Sokolov, N.N. (2013). Recombinant intracellular Rhodospirillum rubrum L-asparaginase with low L-glutaminase activity and antiproliferative effect. Biomeditsinskaia Khimiia, 59(2), 192-208. DOI
  63. Palm, G.J., Lubkowski, J., Derst, C., Schleper, S., Röhm, K.H., Wlodawer, A. (1996) A covalently bound catalytic intermediate in Escherichia coli asparaginase: crystal structure of a Thr-89-Val mutant. FEBS letters, 390(2), 211-216. DOI
  64. El-Ghonemy, D. (2014) Microbial amidases and their industrial applications: A review. Journal of Medical Microbiology and Diagnosis, 4, 1-6. DOI
  65. Borek, D., Kozak, M., Pei, J., Jaskolski, M. (2014) Crystal structure of active site mutant of antileukemic L-asparaginase reveals conserved zinc-binding site. The FEBS journal, 81(18), 4097-4111. DOI
  66. Nguyen, H.A., Su, Y., Lavie, A. (2016) Design and characterization of Erwinia chrysanthemi L-asparaginase variants with diminished L-glutaminase activity. The Journal of biological chemistry, 291(34), 17664-17676. DOI
  67. Nguyen, H.A, Su, Y., Lavie, A. (2016) Structural insight into substrate selectivity of Erwinia chrysanthemi L-asparaginase. Biochemistry, 55(8), 1246- 1253. DOI
  68. Nguyen, H.A., Durden, D.L., Lavie, A. (2017) The differential ability of asparagine and glutamine in promoting the closed/active enzyme conformation rationalizes the Wolinella succinogenes L-asparaginase substrate specificity. Scientific reports, 7, 41643. DOI
  69. Lubkowski, J., Wlodawer, A. (2019) Geometric considerations support the double-displacement catalytic mechanism of L-asparaginase. Protein science: a publication of the Protein Society, 28(10), 1850-1864. DOI
  70. Lubkowski, J., Vanegas, J.M., Chan, W.K., Lorenzi, P., Weinstein, J., Sukharev, S., Fushman, D., Rempe, S., Anishkin, A., Wlodawer, A. (2020) Mechanism of catalysis by L-asparaginase. Biochemistry, 59(20), 1927-1945. DOI
  71. Min Yao, Yoshiaki Yasutake, Hazuki Morita, Isao Tanaka. Structure of the type I L-asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii at 2.16 A resolution Acta Crystallographica Section D: Structural Biology (2005) 61(Pt 3):294-301. DOI
  72. Tomar, R., Garg, D.K., Mishra, R., Thakur, A.K., Kundu, B. (2013) N-terminal domain of Pyrococcus furiosus L-asparaginase functions as a nonspecific, stable, molecular chaperone. The FEBS journal, 280(11), 2688-2699. DOI
  73. Pritsa, A.A., Kyriakidis, D.A. (2001) L-asparaginase of Thermus thermophilus: Purification, properties and identification of essential amino acids for its catalytic activity. Molecular and cellular biochemistry, 216 (1-2), 93-101. DOI
  74. Derst, C., Henseling, J., Röhm, K.H. (1992) Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis. Protein engineering, 5(8), 785-789. DOI
  75. Derst, C., Henseling, J., Röhm, K.H. (2000) Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248. Protein science: a publication of the Protein Society, 9(10), 2009-2017. DOI
  76. Derst, C., Wehner, A., Specht, V., Röhm, K.H. (1994) States and functions of tyrosine residues in Escherichia coli asparaginase II. European journal of biochemistry, 224(2), 533-540. DOI
  77. Bansal, S., Srivastava, A., Mukherjee, G., Pandey, R., Verma, A.,K. Mishra, P., Kundu, B. (2012) Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 26(3), 1161-1171. DOI
  78. Offman, M.N., Krol, M., Patel, N., Krishnan, S., Liu, J., Saha, V., Bates, P.A. (2011) Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity. Blood. 117(5), 1614-1621. DOI
  79. Costa, I.M., Schultz, L., de Araujo Bianchi, P.B., Leite, M.S., Farsky, S.H., de Oliveira, M.A., Pessoa, A., Monteiro, G. (2016) Recombinant L-asparaginase ׀ from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity. Scientific reports, 6(1), 36239. DOI
  80. Karamitros, C.S., Konrad, M. (2014) Bacterial co-expression of the α and β protomers of human L-asparaginase-3: Achieving essential N-terminal exposure of a catalytically critical threonine located in the β-subunit. Protein expression and purification, 93, 1-10. DOI
  81. Karamitros, C.S., Konrad, M. (2014) Human 60-kDa lysophospholipase contains an N-terminal L-asparaginase domain that is allosterically regulated by L-asparagine. The Journal of biological chemistry, 289(19), 12962-12975. DOI
  82. Maqsood, B., Basit. A., Khurshid, M., Bashir, Q. (2020) Characterization of a thermostable, allosteric L-asparaginase from Anoxybacillus flavithermus. International journal of biological macromolecules, 152, 584-592. DOI
  83. Mihooliya, K.N., Nitika, N., Bhambure, R., Rathore, A. (2022) Post-refolding stability considerations for optimization of in-vitro refolding: L-asparaginase as a case study. Biotechnology journal, 18(4), 2200505. DOI
  84. Schymkowitz, J., Borg, J., Stricher, F., Nys, R., Rousseau, F., Serrano, L. (2005) The FoldX web server: an online force field. Nucleic acids research, 33(Web Server issue):W382-8. DOI
  85. Dastmalchi, M., Alizadeh, M., Jamshidi-Kandjan, O., Rezazadeh, H., Hamzeh-Mivehroud, M., Farajollahi, M.M., Dastmalchi, S. (2023) Expression and biological evaluation of an engineered recombinant L-asparaginase designed by In Silico method based on sequence of the enzyme from Escherichia coli. Advanced pharmaceutical bulletin, 13(4), 827-836. DOI
  86. Goyal, G., Bhatt, V.R. (2015) L-asparaginase and venous thromboembolism in acute lymphocytic leukemia. Future oncology (London, England), 11(17), 2459-2470. DOI
  87. Schmiegelow, K., Attarbaschi, A., Barzilai, S., Escherich, G., Frandsen, T., Halsey, C.,Hough, R., Jeha, S., Kato, M., Liang, D.C., Mikkelsen, T.S., Möricke, A., Niinimäki, R., Piette, C., Putti, M.C., Raetz, E., Silverman, L.B., Skinner, R., Tuckuviene, R., van der Sluis, I., Zapotocka, E. (2016) Consensus definitions of 14 severe acute toxic effects for childhood lymphoblastic leukaemia treatment: A delphi consensus. The Lancet. Oncology, 17 (6), e231–e239. DOI
  88. Zhang, Z.X., Nong, F.T., Wang, Y.Z, Yan, C.-X., Gu, Y., Song, P., Sun, X.M. (2022) Strategies for efficient production of recombinant proteins in Escherichia coli: alleviating the host burden and enhancing protein activity. Microbial Cell Factories, 21(1), 191. DOI
  89. Zhang, S., Sun, Y., Zhang, L., Zhang, F., Gao, W. (2023) Thermoresponsive polypeptide fused L-asparaginase with mitigated immunogenicity and enhanced efficacy in treating hematologic malignancies. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 10(23):e2300469. DOI
  90. Zhang, W., Dai, Q., Huang, Z., Xu, W. (2023) Identiication and thermostability modification of the mesophilic L-asparaginase from Limosilactobacillus secaliphilus. Applied biochemistry and biotechnology, 196(6), 1-15. DOI
  91. Kishore, V., Nishita, K.P., Manonmani, H.K. (2015) Cloning, expression and characterization of L-asparaginase from Pseudomonas fluorescens for large scale production in E. coli BL21. 3 Biotech. 5(6), 975-981. DOI
  92. Wang, Y., Xu, W., Wu, H., Zhang, W., Guang, C., Mu, W. (2021) Microbial production, molecular modification, and practical application of L-Asparaginase: A review. International journal of biological macromolecules, 186, 975-983. DOI
  93. Pokrovskaya, M.V., Aleksandrova, S.S., Pokrovsky, V.S., Omeljanjuk, N.M., Borisova A.A., Anisimova, N.Y., Sokolov, N.N. (2012) Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase. Protein expression and purification, 82(1), 150-154. DOI
  94. Maggi, M., Mittelman, S.D., Parmentier, J.H., Colombo, G., Meli, M., Whitmire, J.M., Merrell, D.S., Whitelegge, J., Scotti, C. (2017) A proteaseresistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro. Scientific reports, 7(1), 14479. DOI
  95. Mahboobi, M., Salmanian, A.H., Sedighian, H., Bambai, B. (2023) Molecular modeling and optimization of type II E.coli L-asparginase activity by in silico design and in vitro site-directed mutagenesis. The protein journal, 42(6), 664-674. DOI
  96. Mahboobi, M., Sedighian, H., Hedayati, M., Bambai, B., Saeed, E., Soofian, A.J. (2017) Applying bioinformatic tools for modeling and modifying type II E.coli L-asparginase to present a better therapeutic agent/drug for acute lymphoblastic leukemia. International Journal of Cancer Management, 10(3), e5785. DOI
  97. Ln, R., Doble, M., Rekha, V.P., Pulicherla, K.K. (2011) In silico engineering of L-asparaginase to have reduced glutaminase side activity for effective treatment of acute lymphoblastic leukemia. Journal of pediatric hematology/ oncology, 33(8), 617-621. DOI
  98. Ardalan, N., Akhavan, S.A., Khavari-Nejad, R. (2021) Development of Escherichia coli asparaginase II for the treatment of acute lymphocytic leukemia: in silico reduction of asparaginase II side effects by a novel mutant (V27F). Asian Pacific journal of cancer prevention: APJCP, 22(4), 1137-1147. DOI
  99. Song, Z., Zhang, Q., Wu, W., Pu, Z., Yu, H. (2023) Rational design of enzyme activity and enantioselectivity. Frontiers in bioengineering and biotechnology, 11, 1129149. DOI
  100. Korendovych, I.V. (2018) Rational and semirational protein design. Methods in molecular biology, 1685, 15-23. DOI
References 101 to 595 are available in the PDF version of the article.