Optimasi Waktu Induksi dalam Mengekspresikan Gen Proinsulin secara Intraseluler Menggunakan Inang Pichia pastoris
DOI:
https://doi.org/10.26740/jrba.v2n1.p26-35Keywords:
Proinsulin, Pichia pastoris, Diabetes, Ekspresi, IntraselulerAbstract
Gen proinsulin telah berhasil diinsersi pada galur Pichia pastoris X33, GS115 dan KM71H pada penelitian sebelumnya, namun masih memerlukan optimasi. Penelitian ini dilakukan untuk mengetahui ekspresi galur P. pastoris dan waktu induksi yang optimal dalam mengekspresikan proinsulin. Ekspresi proinsulin dilakukan pada suhu 20 °C selama 120 jam untuk pemilihan galur terbaik. Variasi lama induksi yang digunakan adalah 0, 24, 48, 72, 96, 120 dan 144 jam untuk mengetahui waktu induksi optimal. Kultur kemudian dilisis, dielektroforesis menggunakan Tricine SDS PAGE dan divisualisasikan dengan pewarnaan perak. Berdasarkan ketebalan pita yang terbentuk pada gel elektroforesis, galur X33-X2 menghasilkan proinsulin terbanyak dibandingkan GS115-G11 dan KM71H-K4.. Ekspresi proinsulin optimal pada 120 jam dan mengalami penurunan proinsulin pada waktu induksi 144 jam akibat akumulasi metanol. Galur X33-X2 merupakan inang terbaik dan waktu induksi 120 jam merupakan waktu induksi optimal dalam mengekspresikan proinsulin secara intraseluler. Hasil ini diharapkan dapat menjadi referensi dalam mengembangkan produksi insulin di Indonesia.
References
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Cereghino, J. L., & Cregg, J. M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24(1), 45-66. doi: https://doi.org/10.1111/j.1574-6976.2000.tb00532.x.
Chauhan, A. K., Arora, D., & Khanna, N. (1999). A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris. Process Biochemistry, 34(2), 139-145. Diakses dari http://agris.fao.org/agris-search/search.do?recordID=US201302911699.
Cos, O., Ramon, R., Montesinos, J.L.,& Valero F. (2006). Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review. Microbial Cell Factories, 5(17), 1-20. doi: 10.1186/1475-2859-5-17
Cregg, J. M., Barringer, K. J., Hessler, A. Y., & Madden, K. R. (1985). Pichia pastoris as a host system for transformations. Molecular and Celluler Biology, 5(12), 3376-3385. doi: 10.1128/mcb.5.12.3376.
Cregg, J. M., Tschopp, J. F., Stillman, C., Siegel, R., Akong, M., Craig, W. S., Buckholz, R. G., Madden, K. R., Kellaris, P. A., & Davis, G. R. (1987). High-Level Expression and Efficient Assembly of Hepatitis-B Surface-Antigen in the Methylotrophic Yeast, Pichia pastoris. Nature Biotechnology, 5, 479485. Diakses dari https://www.nature.com/articles/nbt0587-479.
Chiruvolu, V., Cregg, J. M., Meagher, M. M. 1997. Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fed-batch fermentations. Enzyme and Microbiology Technology, 21(4), 277283. doi: https://doi.org/10.1016/S0141-0229(97)00042-2.
Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L., Yansura, D. G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K., & Riggs, A. D. (1979). Expression in Escherichia coli of chemically synthesized genes for human insulin. Proceedings of the National Academy of Science, 76(1), 106-110. doi: 10.1073/pnas.76.1.106
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Gurramkonda, C., Polez, S., Skoko, N., Adnan, A., Gäbel, T., Chugh, D., & Rinas, U. (2010). Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microbial Cell Factories, 9(31), 1-11. doi: 10.1186/1475-2859-9-31.
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Hilt., W., & Wolf, D. H. (1992). Stress induced proteolysis in yeast. Molecular Microbiology, 6 (17), 2437-2442. doi:https://doi.org/10.1111/j.1365-2958.1992.tb01419.x.
International Diabetes Federation (IDF). (2018). IDF Western Pasific Members. Diakses pada 23 November 2018. https://www.idf.org.
Inan, M., & Meagher, M. M. (2001). Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. Journal of Bioscience and Bioengginering. 92(6), 585-589. doi: 10.1016/S1389-1723(01)80321-2
Invitrogen. (2010). EasySelectTM Pichia Expression Kit: For Expression of Recombinant Proteins Using pPICZ and pPICZα in Pichia pastoris. Thermo Scientific.
Kharroubi, A. T., & Darwish, H. M. (2015). Diabetes mellitus: The epidemic of the century. World Journal Diabetes, 6(6), 850-867. doi: 10.4239/wjd.v6.i6.850.
Krainer, F. W., Dietzsch, C., Hajek, T., Herwig, C., Spadiut, O., & Glieder, A. (2012). Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microbiology Cell Factories, 11(22), 1-14. doi: 10.1186/1475-2859-11-22.
Lilly, E. (2018). Humulin. Eli Lilly and Company: Indianapolis.
Matsumoto, H., Hanlu, H., & Komori, N. (2018). Determination of Protein Molecular Weights on SDS-PAGE. Electrophoretic Separation of Proteins, 1855, 101-105. Diakses dari https://link.springer.com/protocol/10.1007/978-1-4939-8793-1_10.
Macauley-Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast, 22(4), 249270. doi: 10.1002/yea.1208
Moore, H. H., Walker, M. D., Lee, F., Kelly, R. B. 1983. Expressing a Human Proinsulin cDNA in a Mouse ACTH-Secreting Cell. Intracellular Storage, Proteolytic Processing, and Secretion on Stimulation. Cell, 35(2), 531-538. doi: 10.1016/0092-8674(83)90187-3.
Nakashima, H., & Nishikawa, K. (1994). Discrimination of Intracellular and Extracellular Proteins Using Amino Acid Composition and Residue-pair Frequencies. Journal Molecular Biology, 238(1), 54-61. Diakses dari https://www.jsbi.org/pdfs/journal1/GIW94/Poster/GIW94P18.pdf.
Philipson, L. H., Bell, G., & Polonsky, K. S. (2015). Donald F. Steiner MD, 1930-2014: Discoverer of proinsulin. Restropective,. 112(4), 940-941. doi: https://doi.org/10.1073/pnas.1423774112.
Steiner, D. F., Peterson, J. D., Tager, H., Emdin, S., Ostberg, & Y. Falkmer, S. 1973. Comparative Aspects of Proinsulin and Insulin Structure and Biosynthesis. Integrative and Comparative Biology, 13(3), 591-604. doi: https://doi.org/10.1093/icb/13.3.591.
Tokarz, V. L., MacDonald, P. E., Klip, A. 2018. The cell biology of systemic insulin function. Journal of Cell Biology, 217(7), 2273-2289. doi: 10.1083/jcb.201802095.
Martius, E., Triyadi, A., Yustika D., & Mahsunah, A. H. (2019). Pengaruh Variasi Konsentrasi Metanol dan Lama Induksi terhadap Ekspresi Proinsulin oleh Pichia pastoris secara Intraseluler. Jurnal Bioteknologi dan Biosains Indonesia 6(1): 93-105. doi: http://dx.doi.org/10.29122/jbbi.v6i1.3176
Vassileva, A., Chugh, D. A., Swaminathan, S., & Khanna, N. (2001). Effect of Copy Number on the Expression Levels of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris. Protein Expression and Purification, 21(1), 71-80. doi: 10.1006/prep.2000.1335.
World Health Organization (WHO). (2016). Global Report on Diabetes. WHO: Geneva.
Yin, H., Liu, Z., Zhang, A., Zhang, T., Luo, J., Shen, J., Chen, L., Zhou, B., Fu, X., Fu, C., & Zhang, Z. (2012). Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris. Gene, 504(1): 122-126. doi: 10.1016/j.gene.2012.04.073.
Zahid, A., Jamil, W., & Begum, R. (2014). Method Development and Validation of SDS-PAGE for Quality Control Testing of Pegylated Interferon Alpha-2a. IOSR Journal of Pharmacy and Biological Sciences, 9(6), 32-36. doi: 10.9790/3008-09643236.
Zhan, C., Yang, Y., Zhang, Z., Li, X., Liu, X., & Bai, Z. (2017). Transcription factor Mxr1 promotes the expression of Aox1 by repressing glycerol transporter 1 in Pichia pastoris. FEMS Yeast Res, 17(4), 1-10. doi: 10.1093/femsyr/fox015.
Zhang W. H., Potter K. J. H., Plantz B. A., Schlegel V. L., Smith L. A., & Meagher M. M. (2003). Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. Journal of Industrial Microbiology and Biotechnology, 30(4), 210-215. doi: 10.1007/s10295-003-0035-3
Bohannon, N. J. V. (1983). Newer Forms of Insulin. The Western Journal of Medicine, 139(1), 115-116. Diakses dari https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1010916/.
Cereghino, J. L., & Cregg, J. M. (2000). Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24(1), 45-66. doi: https://doi.org/10.1111/j.1574-6976.2000.tb00532.x.
Chauhan, A. K., Arora, D., & Khanna, N. (1999). A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris. Process Biochemistry, 34(2), 139-145. Diakses dari http://agris.fao.org/agris-search/search.do?recordID=US201302911699.
Cos, O., Ramon, R., Montesinos, J.L.,& Valero F. (2006). Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review. Microbial Cell Factories, 5(17), 1-20. doi: 10.1186/1475-2859-5-17
Cregg, J. M., Barringer, K. J., Hessler, A. Y., & Madden, K. R. (1985). Pichia pastoris as a host system for transformations. Molecular and Celluler Biology, 5(12), 3376-3385. doi: 10.1128/mcb.5.12.3376.
Cregg, J. M., Tschopp, J. F., Stillman, C., Siegel, R., Akong, M., Craig, W. S., Buckholz, R. G., Madden, K. R., Kellaris, P. A., & Davis, G. R. (1987). High-Level Expression and Efficient Assembly of Hepatitis-B Surface-Antigen in the Methylotrophic Yeast, Pichia pastoris. Nature Biotechnology, 5, 479485. Diakses dari https://www.nature.com/articles/nbt0587-479.
Chiruvolu, V., Cregg, J. M., Meagher, M. M. 1997. Recombinant protein production in an alcohol oxidase-defective strain of Pichia pastoris in fed-batch fermentations. Enzyme and Microbiology Technology, 21(4), 277283. doi: https://doi.org/10.1016/S0141-0229(97)00042-2.
Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L., Yansura, D. G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K., & Riggs, A. D. (1979). Expression in Escherichia coli of chemically synthesized genes for human insulin. Proceedings of the National Academy of Science, 76(1), 106-110. doi: 10.1073/pnas.76.1.106
Grinna, L. S., & Tschopp, J. F. (1989). Size distribution and general structural features of N-linked oligosaccharides from the methylotropic yeast Pichia pastoris. Yeast, 5(2), 107-115. doi: 10.1002/yea.320050206.
Gurramkonda, C., Polez, S., Skoko, N., Adnan, A., Gäbel, T., Chugh, D., & Rinas, U. (2010). Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microbial Cell Factories, 9(31), 1-11. doi: 10.1186/1475-2859-9-31.
Hasslacher, M., Schall, M., Hayn, M., Bona, R., Rumbold, K., Lückl, J., Griengl, H., Kohlwein, S. D., & Schwab, H. (1997). High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expression Purification,11(1), 6171. doi: 10.1006/prep.1997.0765.
Hilt., W., & Wolf, D. H. (1992). Stress induced proteolysis in yeast. Molecular Microbiology, 6 (17), 2437-2442. doi:https://doi.org/10.1111/j.1365-2958.1992.tb01419.x.
International Diabetes Federation (IDF). (2018). IDF Western Pasific Members. Diakses pada 23 November 2018. https://www.idf.org.
Inan, M., & Meagher, M. M. (2001). Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. Journal of Bioscience and Bioengginering. 92(6), 585-589. doi: 10.1016/S1389-1723(01)80321-2
Invitrogen. (2010). EasySelectTM Pichia Expression Kit: For Expression of Recombinant Proteins Using pPICZ and pPICZα in Pichia pastoris. Thermo Scientific.
Kharroubi, A. T., & Darwish, H. M. (2015). Diabetes mellitus: The epidemic of the century. World Journal Diabetes, 6(6), 850-867. doi: 10.4239/wjd.v6.i6.850.
Krainer, F. W., Dietzsch, C., Hajek, T., Herwig, C., Spadiut, O., & Glieder, A. (2012). Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microbiology Cell Factories, 11(22), 1-14. doi: 10.1186/1475-2859-11-22.
Lilly, E. (2018). Humulin. Eli Lilly and Company: Indianapolis.
Matsumoto, H., Hanlu, H., & Komori, N. (2018). Determination of Protein Molecular Weights on SDS-PAGE. Electrophoretic Separation of Proteins, 1855, 101-105. Diakses dari https://link.springer.com/protocol/10.1007/978-1-4939-8793-1_10.
Macauley-Patrick, S., Fazenda, M. L., McNeil, B., & Harvey, L. M. (2005). Heterologous protein production using the Pichia pastoris expression system. Yeast, 22(4), 249270. doi: 10.1002/yea.1208
Moore, H. H., Walker, M. D., Lee, F., Kelly, R. B. 1983. Expressing a Human Proinsulin cDNA in a Mouse ACTH-Secreting Cell. Intracellular Storage, Proteolytic Processing, and Secretion on Stimulation. Cell, 35(2), 531-538. doi: 10.1016/0092-8674(83)90187-3.
Nakashima, H., & Nishikawa, K. (1994). Discrimination of Intracellular and Extracellular Proteins Using Amino Acid Composition and Residue-pair Frequencies. Journal Molecular Biology, 238(1), 54-61. Diakses dari https://www.jsbi.org/pdfs/journal1/GIW94/Poster/GIW94P18.pdf.
Philipson, L. H., Bell, G., & Polonsky, K. S. (2015). Donald F. Steiner MD, 1930-2014: Discoverer of proinsulin. Restropective,. 112(4), 940-941. doi: https://doi.org/10.1073/pnas.1423774112.
Steiner, D. F., Peterson, J. D., Tager, H., Emdin, S., Ostberg, & Y. Falkmer, S. 1973. Comparative Aspects of Proinsulin and Insulin Structure and Biosynthesis. Integrative and Comparative Biology, 13(3), 591-604. doi: https://doi.org/10.1093/icb/13.3.591.
Tokarz, V. L., MacDonald, P. E., Klip, A. 2018. The cell biology of systemic insulin function. Journal of Cell Biology, 217(7), 2273-2289. doi: 10.1083/jcb.201802095.
Martius, E., Triyadi, A., Yustika D., & Mahsunah, A. H. (2019). Pengaruh Variasi Konsentrasi Metanol dan Lama Induksi terhadap Ekspresi Proinsulin oleh Pichia pastoris secara Intraseluler. Jurnal Bioteknologi dan Biosains Indonesia 6(1): 93-105. doi: http://dx.doi.org/10.29122/jbbi.v6i1.3176
Vassileva, A., Chugh, D. A., Swaminathan, S., & Khanna, N. (2001). Effect of Copy Number on the Expression Levels of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris. Protein Expression and Purification, 21(1), 71-80. doi: 10.1006/prep.2000.1335.
World Health Organization (WHO). (2016). Global Report on Diabetes. WHO: Geneva.
Yin, H., Liu, Z., Zhang, A., Zhang, T., Luo, J., Shen, J., Chen, L., Zhou, B., Fu, X., Fu, C., & Zhang, Z. (2012). Intracellular expression and purification of the Canstatin-N protein in Pichia pastoris. Gene, 504(1): 122-126. doi: 10.1016/j.gene.2012.04.073.
Zahid, A., Jamil, W., & Begum, R. (2014). Method Development and Validation of SDS-PAGE for Quality Control Testing of Pegylated Interferon Alpha-2a. IOSR Journal of Pharmacy and Biological Sciences, 9(6), 32-36. doi: 10.9790/3008-09643236.
Zhan, C., Yang, Y., Zhang, Z., Li, X., Liu, X., & Bai, Z. (2017). Transcription factor Mxr1 promotes the expression of Aox1 by repressing glycerol transporter 1 in Pichia pastoris. FEMS Yeast Res, 17(4), 1-10. doi: 10.1093/femsyr/fox015.
Zhang W. H., Potter K. J. H., Plantz B. A., Schlegel V. L., Smith L. A., & Meagher M. M. (2003). Pichia pastoris fermentation with mixed-feeds of glycerol and methanol: growth kinetics and production improvement. Journal of Industrial Microbiology and Biotechnology, 30(4), 210-215. doi: 10.1007/s10295-003-0035-3
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2020-03-31
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Martius, E., Laoditta, F., Sofia, D. Y., & Mahsunah, A. H. (2020). Optimasi Waktu Induksi dalam Mengekspresikan Gen Proinsulin secara Intraseluler Menggunakan Inang Pichia pastoris. Jurnal Riset Biologi Dan Aplikasinya, 2(1), 26–35. https://doi.org/10.26740/jrba.v2n1.p26-35
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