Abstract
Lower induction temperature and polyoxyethylene sorbitan monolaurate (Tween-20) were successfully used to inhibit the aggregation of recombinant human consensus interferon-α mutant (cIFN) during Pichia pastoris fermentation. When the induction temperature was decreased from 30 to 20°C, the cIFN secreted into the medium was in the form of monomers instead of aggregates. The maximum specific activity at 20°C was 4.04 times as high as that at 30°C. There was no obvious effect on the cell growth at 20°C, but the total protein level was decreased. Similar inhibition effect on cIFN aggregation was observed when 0.2 g l−1 Tween-20 was added during induction. Furthermore, there was a synergistic effect found between induction temperature and Tween-20 on the inhibition of cIFN aggregation. The maximum specific activity with Tween-20 at 20°C was 19.9-fold higher than that without Tween-20 at 30°C.
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References
Alton K, Stabinsky Y, Richards R, Ferguson B, Goldstein L, Altrock B, Miller L, Stebbing N (1983) Production, characterization and biological effects of recombinant DNA derived human IFN-α and IFN-γ analogs. In: De Maeyer E, Schellekens H (eds) Biology of the interferon system. Elservier, Amsterdam, pp 119–128
Bitter GA, Chen KK, Banks AR, Lai P-H (1984) Secretion of foreign proteins from Saccharomyces cerevisiae directed by α-factor gene fusions. Proc Natl Acad Sci U S A 81:5330–5334
Curless C, Pope J, Tsai L (1990) Effect of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli. Biotechnol Prog 6:149–152
Daly R, Heart MTW (2005) Expression of heterologous proteins in Pichia pastoris, a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138
Fieschko J, Ritch T (1986) Production of human alpha consensus interferon in recombinant Escherichia coli. Chem Eng Commun 45:229–240
Hao YY, Chu J, Wang YH, Zhang SL, Zhuang YP (2006) Expression and aggregation of recombinant human consensus interferon-α mutant by Pichia pastoris. Biotechnol Lett 28(12):905–909
Hong F, Meinander NQ, Jönsson LJ (2002) Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. Biotechnol Bioeng 79:438–449
Hou YD (1985) Principle and methods of viral genetic engineering. People’s Health Press, Beijing (in Chinese)
Huang SL, Lin FY, Yang CP (2005) Microcalorimetric studies of the effects on the interactions of human recombinant interferon-alpha 2a. Eur J Pharm Sci 24(5):545–552
Jahic M, Gustavsson M, Jansen A-K, Martinelle M, Enfors S-O (2003) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. J Biotechnol 102:45–53
Kreilgaard L, Jones LS, Randolph TW, Frokhaer R, Flink JM, Manning MC, Carpenter JF (1998) Effect of Tween 20 on freeze- thawing- and agitation-induced aggregation of recombinant human factor XIII. J Pharm Sci 87:1597–1603
Liu ZM, Zhao HL, Xue C, Yang SZ, Wu KH (2002) Expression and production of human consensus interferon-α mutant. Chinese Patent CN1435486A, pp 33 (in Chinese)
Klein ML, Bartley TD, Lai PH, Lu HS (1988) Structural characterization of recombinant consensus interferon-α. J Chromatogr 454:205–216
Macauley-Patrick S, Fazenda ML, Mcneil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270
Ozes ON, Reiter Z, Kelin S, Blatt LM, Taylor MW (1992) A comparison of interferon-Con1 with natural recombinant interferon-α: antiviral, antiproliferative and natural killer inducing activities. J Interf Res 12:55–59
Sinha J, Plantz B, Inan M, Meagher MM (2005) Causes of proteolytic degradation of secreted recombinant proteins produced in methylotrophic yeast Pichia pastoris: case study with recombinant ovine interferon-τ. Biotechnol Bioeng 89:102–112
Stratton J, Chiruvolu V, Meagher M (1998) High cell-density fermentation. In: Higgins DR, Cregg JM (eds) Methods in molecular biology, vol. 103. Humana, Totowa, NJ, pp 107–120
Villaverde A, Carrió MM (2003) Protein aggregation in recombinant bacteria: biological role of inclusion bodies. Biotechnol Lett 25:1385–1395
Wang W (1999) Instability, stabilization, and formulation of liquid protein pharmaceuticals. Int J Pharm 185:129–188
Wang JZ, Fan M (2000) Manual of protein technique. Science Press, Beijing (in Chinese)
Wang HH, Gao BY, Chen PL, Dong L, Li YY (1995) Analysis of heterogeneity of gene products (interferon) expressed in yeast. Sci China Ser B Chem Life Sci Earth Sci 38:954–962
Yu CM, Fu L, Qi LQ, Zhang XP, Yu T, Lai DZ, Wang HT, Chen W (2003) Expression, purification and activity analysis of consensus interferon mutant produced in Pichia pastoris. Chin J Biochem Mol Biol 19:3l7–320
Acknowledgements
This research was supported by the National High Technology Research and Development Program of China (863 Program, nos. 2002AA217021 and 2002AA2Z3451). We thank Zhimin Liu for giving P. pastoris GS115/pPIC9-cIFN cells; Kanghua Wu, Yun He, and Qiqi Shi for technical assistance in fermentation and electrophoresis analysis; and Fang Liu for a critical revision of the manuscript.
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Hao, Y., Chu, J., Wang, Y. et al. The inhibition of aggregation of recombinant human consensus interferon-α mutant during Pichia pastoris fermentation. Appl Microbiol Biotechnol 74, 578–584 (2007). https://doi.org/10.1007/s00253-006-0715-9
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DOI: https://doi.org/10.1007/s00253-006-0715-9