Abstract
Human interleukin-3 (hIL-3) is a therapeutically important cytokine involved in the maturation and differentiation of various cells of the immune system. The codon-optimized hIL-3 gene was cloned in fusion with the N-terminus α-mating factor signal peptide of Saccharomyces cerevisiae under an inducible alcohol oxidase 1 (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. A Zeocin concentration up to 2000 mg/L was used to select hyper-producers. The shake flask cultivation studies in the Pichia pastoris GS115 host resulted a maximum recombinant hIL-3 expression level of 145 mg/L in the extracellular medium under the control of AOX1 promoter. The batch fermentation strategy allowed us to attain a fairly pure glycosylated hIL-3 protein in the culture supernatant at a final concentration of 475 mg/L with a high volumetric productivity of 4.39 mg/L/h. The volumetric product concentration achieved at bioreactor level was 3.28 folds greater than the shake flask results. The 6x His-tagged protein was purified using Ni–NTA affinity chromatography and confirmed further by western blot analysis using anti-6x His tag antibody. The glycosylation of recombinant hIL-3 protein was confirmed in a PNGase F deglycosylation reaction where it showed a molecular weight band pattern similar to E. coli produced non-glycosylated hIL-3 protein. The structural properties of recombinant hIL-3 protein were confirmed by CD and fluorescence spectroscopy where protein showed 40 % α-helix, 12 % β-sheets with an emission maxima at 343 nm. MALDI-TOF-TOF analysis was used to establish the protein identity. The biological activity of purified protein was confirmed by the human erythroleukemia TF-1 cell proliferation assay.
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Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12):5301–5317
Bai J, Swartz DJ, Protasevich II, Brouillette CG, Harrell PM, Hildebrandt E, Gasser B, Mattanovich D, Ward A, Chang G, Urbatsch IL (2011) A gene optimization strategy that enhances production of fully functional P-glycoprotein in Pichia pastoris. PLoS One 6(8):e22577
Boysen S, Fogh-Schultz B, Andersen I, Højrup P, Iversen JJ, Wittenhagen P, Nielsen EH, Svehag SE (2004) Recombinant human serum amyloid P component from Pichia pastoris: production and characterization. Protein Expr Purif 35(2):284–292
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Bretthauer RK, Castellino FJ (1999) Glycosylation of Pichia pastoris derived proteins. Biotechnol Appl Biochem 30(Pt3):193–200
Broughton SE, Dhagat U, Hercus TR, Nero TL, Grimbaldeston MA, Bonder CS, Lopez AF, Parker MW (2012) The GM-CSF/IL-3/IL-5 cytokine receptor family: from ligand recognition to initiation of signaling. Immunol Rev 20250(1):277–302
Celik E, Calık P (2012) Production of recombinant proteins by yeast cells. Biotechnol Adv 30(5):1108–1118
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24(1):45–66
Curtis BM, Williams DE, Broxmeyer HE, Dunn J, Farrah T, Jeffery E, Clevenger W, Deroos P, Martin U, Friend D, Craig V, Gayle R, Price R, Cosman D, March CJ, Park LS (1991) Enhanced hematopoietic activity of a human granulocyte/macrophage colony-stimulating factor-interleukin 3 fusion protein. Proc Natl Acad Sci USA 88:5809–5813
Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18(2):119–138
Dexter TM, Spooncer E (1987) Growth and differentiation in the hemopoietic system. Annu Rev Cell Biol 3:423–441
Ding H, Griesel C, Nimtz M, Conradt HS, Weich HA, Jäger V (2003) Molecular cloning, expression, purification, and characterization of soluble full-length, human interleukin-3 with a baculovirus–insect cell expression system. Protein Expr Purif 31(1):34–41
Freeman JJ, Parr GR, Hecht RI, Morris JC, Mckearn JP (1991) Secondary Structure of Human interleukin-3. Inr J Biochem 23(3):353–360
Hermanrud CE, Pathiraja V, Matar A, Duran-Struuck R, Crepeau RL, Srinivasan S, Sachs DH, Huang CA, Wang Z (2012) Expression and purification of non-N-glycosylated porcine interleukin 3 in yeast Pichia pastoris. Protein Expr Purif 82(1):70–74
Higgins DR, Busser K, Comiskey J, Whittier PS, Purcell TJ, Hoeffler JP (1998) Small vectors for expression based on dominant drug resistance with direct multicopy selection. Method Mol Biol 103:41–53
Hohenblum H, Gasser B, Maurer M, Borth N, Mattanovich D (2004) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85(4):367–375
Ihle JN, Pepersack L, Rebar L (1981) Regulation of T cell differentiation: in vitro induction of 20 alpha-hydroxysteroid dehydrogenases in splenic lymphocytes from athymic mice by a unique lymphokine. J Immunol 126(6):2184–2189
Inan M, Aryasomayajula D, Sinha J, Meagher MM (2006) Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase. Biotechnol Bioeng 93(4):771–778
Jahic M, Veide A, Charoenrat T, Teeri T, Enfors SO (2006) Process technology for production and recovery of heterologous proteins with Pichia pastoris. Biotechnol Prog 22(6):1465–1473
Karaoglan M, Yildiz H, Inan M (2014) Screening of signal sequences for extracellular production of Aspergillus niger xylanase in Pichia pastoris. Biochem Eng J 92:16–21
Khasa YP, Khushoo A, Tapryal S, Mukherjee KJ (2011) Optimization of human Granulocyte Macrophage colony stimulating factor (hGM-CSF) expression using native asparaginase and xylanase gene’s signal sequences in Escherichia coli. Appl Biochem Biotechnol 165:523–537
Kim S, d’Anjou M, Lanz KJ, Evans CE, Gibson ER, Olesberg JT, Mallem M, Shandil I, Nylen A, Koerperick EJ, Cooley DW, Brower GA, Small GW, Arnold MA (2015) Real-time monitoring of glycerol and methanol to enhance antibody production in industrial Pichia pastoris bioprocesses. Biochem Eng J 94:115–124
Kitamura T, Tange T, Terasawa T, Chiba S, Urabe A, Takaku F (1989) Establishment and characterization of a unique human cell line that proliferates dependently on CM-CSF, IL-3, or erythropoietin. J Cell Physio 40:323–334
Klein BK, Olins PO, Bauer SC, Caparon MH, Easton AM, Braford SR, Abrams MA, Klover JA, Paik K, Thomas JW, Hood WF, Shieh JJ, Polazzi JO, Donnelly AM, Zeng DL, Welply JK, McKearn JP (1999) Use of combinatorial mutagenesis to select for multiply substituted human interleukin-3 variants with improved pharmacologic properties. Exp Hematol 27(12):1746–1756
Li H, Li N, Gao X, Kong X, Li S, Xu S, Jin AS, Wu D (2011) High level expression of active recombinant human interleukin-3 in Pichia pastoris. Protein Expr Purif 80(2):185–193
Li H, Wang Y, Xu A, Li S, Jin S, Wu D (2011) Large-scale production, purification and bioactivity assay of recombinant human interleukin-6 in the methylotrophic yeast Pichia pastoris. FEMS Yeast Res 11(2):160–167
Liu Y, Wu C, Wang J, Mo W, Yu M (2013) Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris. Appl Microbiol Biotechnol 97(24):10349–10358
Lutsenko LV, Gurevich AI, Ptitsyn LR, Riazanova LA, Smirnov VA (1992) Recombinant interleukin-3 expression system in E. coli. Bioorg Khim 18(3):391–397
Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22(4):249–270
Mangi MH, Newland AC (1999) Interleukin-3 in hematology and oncology: current state of knowledge and future directions. Cytokines Cell Mol Ther 5(2):87–95
Ning D, Junjian X, Xunzhang W, Wenyin C, Qing Z, Kuanyuan S, Guirong R, Xiangrong R, Qingxin L, Zhouyao Y (2003) Expression, purification, and characterization of humanized anti-HBs Fab fragment. J Biochem 134(6):813–817
Pal Y, Khushoo A, Mukherjee KJ (2006) Process optimization of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture. Appl Microbiol Biotechnol 69(6):577–650
Potvin G, Ahmad A, Zhang Z (2012) Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: a review. Biochem Eng J 64:91–105
Sears IB, O’Connor J, Rossanese OW, Glick BS (1998) A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris. Yeast 14(8):783–790
Su Y, Shi-Yan L, Ghosh S, Ortiz J, Hogge DE, Frankel AE (2010) Characterization of variant diphtheria toxin-interleukin-3 fusion protein, DTIL3K116 W, for phase I clinical trials. Biologicals 38(1):144–149
Westers L, Dijkstra DS, Westers H, van Dijl JM, Quax WJ (2006) Secretion of functional human interleukin-3 from Bacillus subtilis. J Biotechnol 123(2):211–224
Wu JM, Lin JC, Chieng LL, Lee CK, Hsu TA (2003) Combined use of GAP and AOX1 promoter to enhance the expression of human granulocyte-macrophage-colony-stimulating factor in Pichia pastoris. Enzyme MicrobTechnol 33(4):453–459
Xie J, Zhang L, Ye Q, Zhou Q, Xin L, Du P, Gan R (2003) Angiostatin production in cultivation of Pichia pastoris fed with mixed carbon sources. Biotechnol Lett 25(2):173–177
Yang YC, Ciarletta AB, Temple PA, Chung MP, Kovacic S, Witek-Giannotti JS, Leary AC, Kriz R, Donahue RE, Wong GG (1986) Human IL-3 (multi-CSF): identification by expression cloning of a novel hematopoietic growth factor related to murine IL-3. Cell 47(1):3–10
Yun SI, Yahya ARM, Cossar D, Anderson WA, Scharer JM, Young MM (2001) Temperature downshift increases recombinant cytokine titer in Streptomyces lividans fermentation. Biotechnol Lett 23:1903–1905
Zhang W, Inan M, Meagher MM (2000) Fermentation strategies for recombinant protein expression in the methylotrophic yeast Pichia pastoris. Biotechnol Bioprocess Eng 5(4):275–287
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This work is supported by Department of Biotechnology (DBT), Government of India, New Delhi (Grant No. BT/PR5822/PID/6/684/2012). Vikas Kumar Dagar, Adivitiya, and Nirmala Devi are the recipients of a senior research fellowship from Indian Council of Medical Research (ICMR), Council of Scientific and Industrial Research (CSIR) and University Grants Commission (UGC) (Govt. of India) respectively.
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Dagar, V.K., Adivitiya, Devi, N. et al. Bioprocess development for extracellular production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris . J Ind Microbiol Biotechnol 43, 1373–1386 (2016). https://doi.org/10.1007/s10295-016-1816-9
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DOI: https://doi.org/10.1007/s10295-016-1816-9