Araştırma Makalesi
BibTex RIS Kaynak Göster

Yıl 2025, Cilt: 43 Sayı: 1, 282 - 289, 28.02.2025

Aysun Özçelik , Fatma Ersöz , Semiramis Yilmaz Burcu Emine Tefon Öztürk , Mehmet İnan

Öz

Kaynakça

  • REFERENCES
  • [1] Kjeldsen T. Yeast secretory expression of insulin precursors. Appl Microbiol Biotechnol 2000;54:277–286. [CrossRef]
  • [2] Ahmad B. Pharmacology of insulin. Br J Diabetes Vasc Dis 2004;4:10–14. [CrossRef]
  • [3] Kjeldsen T, Frost-Petterson A, Hach M. Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 1999;29:79–86. [CrossRef]
  • [4] Gurramkonda C, Polez S, Skoko N, Adnan A, Gabel T, Chugh D, et al. 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. Microb Cell Fact 2010;9:1–11. [CrossRef]
  • [5] Weydemann U, Keup P, Piontek M, Strasser AWM, Schweden J, Gellissen G, et al. High-level secretion hirudin by Hansenula polymorpha-authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 1995;44:377–385. [CrossRef]
  • [6] Gellissen G, Melber K. Methylotrophic yeast Hansenula polymorpha as production organism for recombinant pharmaceuticals. Arzneimittelforschung 1996;46:943–948.
  • [7] Raymond CK, Bukowski T, Holderman SD, Ching AFT, Vanaja E, Stamm MR. Development of the Methylotrophic Yeast Pichia methanolica for the Expression of the 65 Kilodalton Isoform of Human Glutamate Decarboxylase. Yeast 1998;14:11–23. [CrossRef]
  • [8] Hagenson MJ. Production of recombinant proteins in the methylotrophic yeast Pichia pastoris. Bioprocess Technol 1991;12:193–212.
  • [9] Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 2000;24:45–66. [CrossRef]
  • [10] Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system Yeast 2005;22:249–270. [CrossRef]
  • [11] İnan M, Meagher MM. Non-Repressing Carbon Sources for Alcohol Oxidase (AOX1) Promoter of Pichia pastoris. J Biosci Bioeng 2001;92:585–589. [CrossRef]
  • [12] Baumann K, Maurer M, Dragosits M, Cos O, Ferrer P, Mattanovich D. Hypoxic fed-batch cultivation of pichia pastoris ıncreases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 2008;100:177–183. [CrossRef]
  • [13] Hilt W, Wolf DH. Stress-induced proteolysis in yeast. Mol Microbiol 1992;6:2437–2442. [CrossRef]
  • [14] Xiao AF, Zhou XS, Zhou L, Zhang YX. Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation. Appl Microbiol Biotechnol 2006;72:837–844. [CrossRef]
  • [15] Mateles RI. Calculation of oxygen required for cell production. Biotechnol Bioeng 1971;13:315–328. [CrossRef]
  • [16] Lin J, Panigraphy D, Trinh LB, Folkman J, Shiloach J. Production process for recombinant human angiostatin in Pichia pastoris. J Ind Microbiol Biotechnol 2000;24:31–35. [CrossRef]
  • [17] Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 1997;186:37–44. [CrossRef]
  • [18] Goodrick JC, Xu M, Finnegan R, Schilling BM, Schiavi S, Hoppe H, Wan NC. High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris espression system. Biotechnol Bioeng 2001;74:492–497. [CrossRef]
  • [19] Olezdzka G, Dazbrowski S, Kur J. High levels expression, secretion and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris. Protein Expr Purif 2003;29:223–229. [CrossRef]
  • [20] Döring F, Theis S, Daniel H. Use of the glyceraldehydes- 3-phosphate dehydrogenase promoter for production of functional mammalian membrane transport proteins in the yeast Pichia pastoris. Biochem Biophys Res Commun 1998;250:531–535. [CrossRef]
  • [21] Yang L, Dong W, He J, Ren X, Yan W. Expression and purification of natural N-terminal recombinant bovine pancreatic trypsin inhibitor from Pichia pastoris. Biol Pharm Bull 2008;31:1680–1685. [CrossRef]
  • [22] Xiong R, Chen J, Chen J. Secreted expression of human lysozyme in the yeast Pichia pastoris under the direction of the signal peptide from human serum albümin. Biotechnol Appl Biochem 2008;5:129–134. [CrossRef]
  • [23] Tian S, Li Q, Yao W, Xu C. Construction and characterization of a potent, long-lasting recombinant human serum albumin-interferon a1 fusion protein expressed in Pichia pastoris. Protein Exp Purif 2013;90:124–128. [CrossRef]
  • [24] Vassileva A, Chugh DA, Swaminathan S, Khanna N. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expres Purif 2001;21;71–80. [CrossRef]
  • [25] Norden K, Agemark M, Danielson JA, Alexandersson E, Kjellbom P, Johanson U. Increasing gene dosage greatly enhances recombinant expression of aqua porins in Pichia pastoris. BMC Biotechnol 2011;11:1–12. [CrossRef]
  • [26] Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 2004;85:367–375. [CrossRef]
  • [27] Türkanoğlu Özçelik A. Comparison of multicopy pro-microbialtransglutaminase encoded gene expression in Pichia pastoris. Acta Alimentaria 2019;48:384–392. [CrossRef]
  • [28] Shen Q, Wu M, Wang HB, Naranmandura H, Chen SQ. The effect of gene copy number and coexpression of chaperone on production of albumin fusion proteins in Pichia pastoris. Appl Microbiol Biot 2012;96:763–772. [CrossRef]
  • [29] Hohenblum H, Borth N, Mattanovich D. Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J Biotechnol 2003;102:281–290. [CrossRef]
  • [30] Kjeldsen T, Frost-Petterson A, Hach M. Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 1999;29:79–86. [CrossRef]
  • [31] Nurdiani D, Nuruliawaty Utami H, Putro EW, Kusharyoto W. Enhancement in human insulin precursor secretion by Pichia pastoris through modification of expression conditions. Hayati J Biosci 2022;29:22–30. [CrossRef]
  • [32] Utami N, Nurdiani D, Hariyatun H, Putro EW, Patria FP, Kusharyoto W. Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison. J Genetic Eng Biotechnol 2023;21:3–15. [CrossRef]

Applicability of the constitutive GAP promoter for recombinant human insulin hormone production in Pichia pastoris

Yıl 2025, Cilt: 43 Sayı: 1, 282 - 289, 28.02.2025

Aysun Özçelik , Fatma Ersöz , Semiramis Yilmaz Burcu Emine Tefon Öztürk , Mehmet İnan

Öz

Insulin is a hormone regulates glucose homeostasis in the body. In this study, the precursor of the human insulin hormone was expressed under the constitutive Glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The expression vector was transferred into the P. pastoris X33 strain by electroporation. After the protein expression study in 5L scale bioreactor, the samples were analysed by SDS-PAGE and ELISA methods. In this study, 11 mg/ L of protein was produced in the bioreactor at the end of production. The MALDI-TOF analysis confirmed that the protein produced was a precursor of the human insulin hormone. At the end of the study, it can be said that a bioprocess has been developed under bioreactor conditions to
produce human insulin precursors by using constitutive GAP promoter of P. pastoris.

Anahtar Kelimeler

Bioreactor GAP Promoter Human Insulin Pichia Pastoris Recombinant Protein

Kaynakça

  • REFERENCES
  • [1] Kjeldsen T. Yeast secretory expression of insulin precursors. Appl Microbiol Biotechnol 2000;54:277–286. [CrossRef]
  • [2] Ahmad B. Pharmacology of insulin. Br J Diabetes Vasc Dis 2004;4:10–14. [CrossRef]
  • [3] Kjeldsen T, Frost-Petterson A, Hach M. Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 1999;29:79–86. [CrossRef]
  • [4] Gurramkonda C, Polez S, Skoko N, Adnan A, Gabel T, Chugh D, et al. 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. Microb Cell Fact 2010;9:1–11. [CrossRef]
  • [5] Weydemann U, Keup P, Piontek M, Strasser AWM, Schweden J, Gellissen G, et al. High-level secretion hirudin by Hansenula polymorpha-authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 1995;44:377–385. [CrossRef]
  • [6] Gellissen G, Melber K. Methylotrophic yeast Hansenula polymorpha as production organism for recombinant pharmaceuticals. Arzneimittelforschung 1996;46:943–948.
  • [7] Raymond CK, Bukowski T, Holderman SD, Ching AFT, Vanaja E, Stamm MR. Development of the Methylotrophic Yeast Pichia methanolica for the Expression of the 65 Kilodalton Isoform of Human Glutamate Decarboxylase. Yeast 1998;14:11–23. [CrossRef]
  • [8] Hagenson MJ. Production of recombinant proteins in the methylotrophic yeast Pichia pastoris. Bioprocess Technol 1991;12:193–212.
  • [9] Cereghino JL, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 2000;24:45–66. [CrossRef]
  • [10] Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system Yeast 2005;22:249–270. [CrossRef]
  • [11] İnan M, Meagher MM. Non-Repressing Carbon Sources for Alcohol Oxidase (AOX1) Promoter of Pichia pastoris. J Biosci Bioeng 2001;92:585–589. [CrossRef]
  • [12] Baumann K, Maurer M, Dragosits M, Cos O, Ferrer P, Mattanovich D. Hypoxic fed-batch cultivation of pichia pastoris ıncreases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 2008;100:177–183. [CrossRef]
  • [13] Hilt W, Wolf DH. Stress-induced proteolysis in yeast. Mol Microbiol 1992;6:2437–2442. [CrossRef]
  • [14] Xiao AF, Zhou XS, Zhou L, Zhang YX. Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation. Appl Microbiol Biotechnol 2006;72:837–844. [CrossRef]
  • [15] Mateles RI. Calculation of oxygen required for cell production. Biotechnol Bioeng 1971;13:315–328. [CrossRef]
  • [16] Lin J, Panigraphy D, Trinh LB, Folkman J, Shiloach J. Production process for recombinant human angiostatin in Pichia pastoris. J Ind Microbiol Biotechnol 2000;24:31–35. [CrossRef]
  • [17] Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM. Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 1997;186:37–44. [CrossRef]
  • [18] Goodrick JC, Xu M, Finnegan R, Schilling BM, Schiavi S, Hoppe H, Wan NC. High-level expression and stabilization of recombinant human chitinase produced in a continuous constitutive Pichia pastoris espression system. Biotechnol Bioeng 2001;74:492–497. [CrossRef]
  • [19] Olezdzka G, Dazbrowski S, Kur J. High levels expression, secretion and purification of the thermostable aqualysin I from Thermus aquaticus YT-1 in Pichia pastoris. Protein Expr Purif 2003;29:223–229. [CrossRef]
  • [20] Döring F, Theis S, Daniel H. Use of the glyceraldehydes- 3-phosphate dehydrogenase promoter for production of functional mammalian membrane transport proteins in the yeast Pichia pastoris. Biochem Biophys Res Commun 1998;250:531–535. [CrossRef]
  • [21] Yang L, Dong W, He J, Ren X, Yan W. Expression and purification of natural N-terminal recombinant bovine pancreatic trypsin inhibitor from Pichia pastoris. Biol Pharm Bull 2008;31:1680–1685. [CrossRef]
  • [22] Xiong R, Chen J, Chen J. Secreted expression of human lysozyme in the yeast Pichia pastoris under the direction of the signal peptide from human serum albümin. Biotechnol Appl Biochem 2008;5:129–134. [CrossRef]
  • [23] Tian S, Li Q, Yao W, Xu C. Construction and characterization of a potent, long-lasting recombinant human serum albumin-interferon a1 fusion protein expressed in Pichia pastoris. Protein Exp Purif 2013;90:124–128. [CrossRef]
  • [24] Vassileva A, Chugh DA, Swaminathan S, Khanna N. Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Protein Expres Purif 2001;21;71–80. [CrossRef]
  • [25] Norden K, Agemark M, Danielson JA, Alexandersson E, Kjellbom P, Johanson U. Increasing gene dosage greatly enhances recombinant expression of aqua porins in Pichia pastoris. BMC Biotechnol 2011;11:1–12. [CrossRef]
  • [26] Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 2004;85:367–375. [CrossRef]
  • [27] Türkanoğlu Özçelik A. Comparison of multicopy pro-microbialtransglutaminase encoded gene expression in Pichia pastoris. Acta Alimentaria 2019;48:384–392. [CrossRef]
  • [28] Shen Q, Wu M, Wang HB, Naranmandura H, Chen SQ. The effect of gene copy number and coexpression of chaperone on production of albumin fusion proteins in Pichia pastoris. Appl Microbiol Biot 2012;96:763–772. [CrossRef]
  • [29] Hohenblum H, Borth N, Mattanovich D. Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J Biotechnol 2003;102:281–290. [CrossRef]
  • [30] Kjeldsen T, Frost-Petterson A, Hach M. Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 1999;29:79–86. [CrossRef]
  • [31] Nurdiani D, Nuruliawaty Utami H, Putro EW, Kusharyoto W. Enhancement in human insulin precursor secretion by Pichia pastoris through modification of expression conditions. Hayati J Biosci 2022;29:22–30. [CrossRef]
  • [32] Utami N, Nurdiani D, Hariyatun H, Putro EW, Patria FP, Kusharyoto W. Full-length versus truncated α-factor secretory signal sequences for expression of recombinant human insulin precursor in yeast Pichia pastoris: a comparison. J Genetic Eng Biotechnol 2023;21:3–15. [CrossRef]
Toplam 33 adet kaynakça vardır.

Ayrıntılar

Birincil Dil İngilizce
Konular Yapısal Biyoloji
Bölüm Research Articles
Yazarlar

Aysun Özçelik 0000-0003-2537-4220

Fatma Ersöz 0000-0002-9647-1231

Semiramis Yilmaz 0000-0001-7683-7059

Burcu Emine Tefon Öztürk 0000-0003-1690-9879

Mehmet İnan 0000-0003-1806-7927

Yayımlanma Tarihi 28 Şubat 2025
Gönderilme Tarihi 24 Kasım 2023
Kabul Tarihi 26 Mart 2024
Yayımlandığı Sayı Yıl 2025 Cilt: 43 Sayı: 1

Kaynak Göster

Vancouver Özçelik A, Ersöz F, Yilmaz S, Tefon Öztürk BE, İnan M. Applicability of the constitutive GAP promoter for recombinant human insulin hormone production in Pichia pastoris. SIGMA. 2025;43(1):282-9.

IMPORTANT NOTE: JOURNAL SUBMISSION LINK https://eds.yildiz.edu.tr/sigma/