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Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density Pichia pastoris bioreactor cultivations

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DOI: 10.4236/abb.2012.33033    4,844 Downloads   8,552 Views   Citations

ABSTRACT

The methanol inducible alcohol oxidase 1 promoter and the Saccharomyces cerevisiae alpha-factor prepro secretion signal were used to drive expression and secretion of a mucin-type fusion protein by Pichia pastoris in 1 L scale bioreactors. The aim of the study was to understand how varying expression rates influenced the secretion dynamics of the fusion protein in terms of intracellular- and extracellular concentrations. Endoplasmic reticulum (ER) folding stress was assessed by the relative expression of the unfolded protein response controlled KAR2 gene. Three predefined methanol feeding models were applied to control the fusion protein synthesis rate. To track the fusion protein synthesis in a non-invasive manner and to follow its intracellular distribution, its C-terminal was linked to the green fluorescent protein. Under all conditions the fusion protein was found to partially accumulate intracellularly, where the major fraction was an insoluble, fluorescent full-sized protein. The high degree of glycosylation of the insoluble fusion protein indicated a secretory bottle-neck in the Golgi-system. This result was consistent with low ER folding stress as quantified by the relative expression of the KAR2 gene. Reduction of recombinant protein synthesis rate, by using lower feed rates of methanol, enhanced extracellular concentrations from 8 to 18 mg·L–1 and reduced the rate of intracellular accumulation. This clearly demonstrates the importance of tuning the synthesis rate with secretory bottle-necks to maintain secretion.

Conflicts of Interest

The authors declare no conflicts of interest.

Cite this paper

Sjöblom, M. , Lindberg, L. , Holgersson, J. and Rova, U. (2012) Secretion and expression dynamics of a GFP-tagged mucin-type fusion protein in high cell density Pichia pastoris bioreactor cultivations. Advances in Bioscience and Biotechnology, 3, 238-248. doi: 10.4236/abb.2012.33033.

References

[1] Cereghino, J.L. and Cregg, J.M. (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews, 24, 45-66. doi:10.1111/j.1574-6976.2000.tb00532.x
[2] Hohenblum, H., Gasser, B., Maurer, M., Borth, N. and Mattanovich, D. (2004) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnology and Bioengineering, 85, 367-375. doi:10.1002/bit.10904
[3] Resina, D., Cos, O., Ferrer, P. and Valero, F. (2005) Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter. Biotechnology and Bioengineering, 91, 760-767. doi:10.1002/bit.20545
[4] McGrew, J.T., Leiske, D., Dell, B., Klinke, R., Krasts, D., Wee, S.F., Abbott, N., Armitage, R. and Harrington, K. (1997) Expression of trimeric CD40 ligand in Pichia pastoris: Use of a rapid method to detect high-level expressing transformants. Gene, 187, 193-200. doi:10.1016/S0378-1119(96)00747-0
[5] Paifer, E., Margolles, E., Cremata, J., Montesino, R., Herrera, L. and Delgado, J.M. (1994) Efficient expression and secretion of recombinant alpha-amylase in Pichia pastoris using 2 different signal sequences. Yeast, 10, 1415-1419. doi:10.1002/yea.320101104
[6] Zhao, H.L., He, Q., Xue, C., Sun, B., Yao, X.Q. and Liu, Z.M. (2009) Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterization. FEMS Yeast Research, 9, 591-599. doi:10.1111/j.1567-1364.2009.00498.x
[7] Bao, W.G. and Fukuhara, H. (2001) Secretion of human proteins from yeast: Stimulation by duplication of polyubiq- uitin and protein disulfide isomerase genes in Kluyvero-myces lactis. Gene, 272, 103-110. doi:10.1016/S0378-1119(01)00564-9
[8] Mukaiyama, H., Tohda, H. and Takegawa, K. (2010) Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosac-charomyces pombe. Applied Microbiology and Biotechnology, 86, 1135-1143. doi:10.1007/s00253-009-2393-x
[9] Marx, H., Sauer, M., Resina, D., Vai, M., Porro, D., Valero, F., Ferrer, P. and Mattanovich, D. (2006) Cloning, disruption and protein secretory phenotype of the GAS1 homo-logue of Pichia pastoris. FEMS Microbiology Letters, 264, 40-47. doi:10.1111/j.1574-6968.2006.00427.x
[10] Cos, O., Ramon, R., Montesinos, J. L. and 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. doi:10.1186/1475-2859-5-17
[11] Jahic, M., Veide, A., Charoenrat, T., Teeri, T. and Enfors, S.O. (2006) Process technology for production and recovery of heterologous proteins with Pichia pastoris, Bio- technology Progress, 22, 1465-1473.
[12] Jahic, M., Gustavsson, M., Jansen, A.K., Martinelle, M. and Enfors, S.O. (2003) Analysis and control of proteolysis of a fusion protein in Pichia pastoris fed-batch processes. Journal of Biotechnology, 102, 45-53. doi:10.1016/S0168-1656(03)00003-8
[13] Zhang, Y.W., Liu, R.J. and Wu, X.Y. (2007) The proteolytic systems and heterologous proteins degradation in the methylotro-phic yeast Pichia pastoris. Annals of Microbiology, 57, 553-560. doi:10.1007/BF03175354
[14] Huang, D. and Shusta, E.V. (2006) A yeast platform for the production of single-chain antibody-green fluorescent protein fusions. Applied Environmental Microbiology, 72, 7748-7759. doi:10.1128/AEM.01403-06
[15] Kimata, Y., Kimata, Y.L., Shimizu, Y., Abe, H., Farcasanu, R.C., Takeuchi, M., Rose, M.D. and Kohno, K. (2003) Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins. Molecular Biology of the Cell, 14, 2559-2569. doi:10.1091/mbc.E02-11-0708
[16] Schroder, M. and Kaufman, R.J. (2005) The mammalian unfolded protein response. Annual Review of Biochemistry, 74, 739-789. doi:10.1146/annurev.biochem.73.011303.074134
[17] Travers, K.J., Patil, C.K., Wodicka, L., Lockhart, D.J., Weissman, J.S. and Walter, P. (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degra- dation. Cell, 101, 249-258. doi:10.1016/S0092-8674(00)80835-1
[18] Gemmill, T.R. and Trimble, R.B. (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. BBA-General Subjects, 1426, 227-237. doi:10.1016/S0304-4165(98)00126-3
[19] Liu, J.N., Qian, Y.J. and Holgersson, J. (1997) Removal of xenore-active human anti-pig antibodies by absorption on re-combinant mucin-containing glycoproteins carrying the Gal alpha 1,3Gal epitope. Transplantation, 63, 1673- 1682. doi:10.1097/00007890-199706150-00023
[20] Gustavsson, M., Lehtio, J., Denman, S., Teeri, T.T., Hult, K. and Martinelle, M. (2001) Stable linker peptides for a cellu-lose-binding domain-lipase fusion protein expressed in Pichia pastoris. Protein Engineering, 14, 711-715. doi:10.1093/protein/14.9.711
[21] Liu, J.N., Gustafsson, A., Breimer, M.E., Kussak, A. and Holgersson, J. (2005) Anti-pig antibody adsorption effi- cacy of alpha-Gal car-rying recombinant P-selectin glyco- protein ligand-1/immunoglobulin chimeras increases with core 2 beta 1,6-N-acetylglucosaminyltransferase expres- sion. Glycobiology, 15, 571-583. doi:10.1093/glycob/cwi037
[22] Guerfal, M., Ryckaert, S., Jacobs, P.P., Ameloot, P., Van Craenenbroeck, K., Derycke, R. and Callewaert, N. (2010) The HAC1 gene from Pichia pastoris: Characterization and effect of its over expression on the production of secreted, surface displayed and membrane proteins. Microbial Cell Factories, 9, 49. doi:10.1186/1475-2859-9-49
[23] Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods, 25, 402- 408. doi:10.1006/meth.2001.1262
[24] Gustafsson, A. and Holgersson, J. (2006) A new generation of carbohy-drate-based therapeutics: Recombinant mucin-type fusion proteins as versatile inhibitors of protein-carbohydrate interactions. Expert Opinion on Drug Discovery, 1, 161-178. doi:10.1517/17460441.1.2.161
[25] Gustafsson, A., Sjoblom, M., Strindelius, L., Johansson, T., Flecken-stein, T., Chatzissavidou, N., Lindberg, L., Angstrom, J., Rova, U. and Holgersson, J. (2011) Pichia pastoris-produced mucintype fusion proteins with multi-valent O-glycan substitution as targeting molecules for mannose-specific receptors of the immune system. Glycobiology, 21, 1071-1086. doi:10.1093/glycob/cwr046
[26] Tsien, R.Y. (1998) The green fluorescent protein. Annual Review of Biochemistry, 67, 509-544. doi:10.1146/annurev.biochem.67.1.509
[27] Huang, D. and Shusta, E.V. (2006) A yeast platform for the production of single-chain antibody-green fluorescent protein fusions. Applied and Environmental Microbiology, 72, 7748-7759. doi:10.1128/AEM.01403-06
[28] Van den Steen, P., Rudd, P.M., Proost, P., Martens, E., Paemen, L., Kuster, B., van Damme, J., Dwek, R.A. and Opdenakker, G. (1998) Oligosaccharides of recombinant mouse gelatinase B variants. BBA-General Subjects, 1425, 587-598. doi:10.1016/S0304-4165(98)00113-5
[29] Ng, D.T.W., Spear, E.D. and Walter, P. (2000) The unfolded protein response regulates multiple aspects of secretory and membrane protein biogenesis and endoplasmic reticulum quality control. Journal of Cell Biology, 150, 77-88. doi:10.1083/jcb.150.1.77
[30] Bernales, S., Papa, F.R. and Walter, P. (2006) Intracellular signaling by the unfolded protein response. Annual Review of Cell and Developmental Biology, 22, 487-508. doi:10.1146/annurev.cellbio.21.122303.120200
[31] Bevis, B.J., Hammond, A.T., Reinke, C.A. and Glick, B. S. (2002) De novo formation of transitional ER sites and Golgi structures in Pichia pastoris. Nature Cell Biology, 4, 750-756. doi:10.1038/ncb852
[32] Curvers, S., Linnemann, J., Klauser, T., Wandrey, C. and Takors, R. (2001) Recombinant protein production with Pichia pastoris in continous fermentation—Kinetic analysis of growth and product formation. Chemie Ingenieur Technik, 73, 1615-1621. doi:10.1002/1522-2640(200112)73:12<1615::AID-CITE1615>3.3.CO;2-Y

  
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