Use of Human Serum Albumin Fusion Tags for Recombinant Protein Secretory Expression in the Methylotrophic Yeast Hansenula polymorpha

메탄올 자화효모 Hansenula polymorpha에서의 재조합 단백질 분비발현을 위한 인체 혈청 알부민 융합단편의 활용

  • Song, Ji-Hye (Systems and Synthetic Biology Research Center, KRIBB) ;
  • Hwang, Dong Hyeon (Systems and Synthetic Biology Research Center, KRIBB) ;
  • Oh, Doo-Byoung (Systems and Synthetic Biology Research Center, KRIBB) ;
  • Rhee, Sang Ki (Department of Medicinal Biotechnology, Soon Chun Hyang University) ;
  • Kwon, Ohsuk (Systems and Synthetic Biology Research Center, KRIBB)
  • 송지혜 (한국생명공학연구원 바이오합성 연구센터) ;
  • 황동현 (한국생명공학연구원 바이오합성 연구센터) ;
  • 오두병 (한국생명공학연구원 바이오합성 연구센터) ;
  • 이상기 (순천향대학교 의약바이오학과) ;
  • 권오석 (한국생명공학연구원 바이오합성 연구센터)
  • Received : 2012.07.27
  • Accepted : 2012.11.13
  • Published : 2013.03.28


The thermotolerant methylotrophic yeast Hansenula polymorpha is an attractive model organism for various fundamental studies, such as the genetic control of enzymes involved in methanol metabolism, peroxisome biogenesis, nitrate assimilation, and resistance to heavy metals and oxidative stresses. In addition, H. polymorpha has been highlighted as a promising recombinant protein expression host, especially due to the availability of strong and tightly regulatable promoters. In this study, we investigated the possibility of employing human serum albumin (HSA) as the fusion tag for the secretory expression of heterologous proteins in H. polymorpha. A set of four expression cassettes, which contained the methanol oxidase (MOX) promoter, translational HSA fusion tag, and the terminator of MOX, were constructed. The expression cassettes were also designed to contain sequences for accessory elements including His8-tag, $2{\times}(Gly_4Ser_1)$ linkers, tobacco etch virus protease recognition sites (Tev), multi-cloning sites, and strep-tags. To determine the effects of the size of the HSA fusion tag on the secretory expression of the target protein, each cassette contained the HSA gene fragment truncated at a specific position based on its domain structure. By using the Green fluorescence protein gene as the reporter, the properties of each expression cassette were compared in various conditions. Our results suggest that the translational HSA fusion tag is an efficient tool for the secretory expression of recombinant proteins in H. polymorpha.


Hansenula polymorpha;HSA fusion tag;protein secretion;expression system


  1. Agaphonov, M. O., P. M. Trushkina, J. H. Sohn, E. S. Choi, S. K. Rhee, and M. D. Ter-Avanesyan. 1999. Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1. Yeast 15: 541-551.<541::AID-YEA392>3.0.CO;2-G
  2. Bradford, M. M. 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.
  3. Buckholz, R. G. and M. A. Gleeson. 1991. Yeast systems for the commercial production of heterologous proteins. Biotechnology( NY) 9: 1067-1072.
  4. Carlson, M. 1987. Regulation of sugar utilization in Saccharomyces species. J. Bacteriol. 169: 4873-4877.
  5. Chow, T. Y. and J. J. Ash. 1992. Screening and identification of a gene, PSE-1, that affects protein secretion in Saccharomyces cerevisiae. J. Cell. Sci. 101: 709-719.
  6. Damasceno, L. M., C.-Jr Huang, and C. A. Batt. 2012. Protein secretion in Pichia pastoris and advances in protein production. Appl. Microbiol. Bitechnolol. 93: 31-39.
  7. DeJuan, C. and R. Lagunas. 1986. Inactivation of the galactose transport system in Saccharomyces cerevisiae, FEBS Lett. 207: 258-261.
  8. Gatzke, R., U. Weydemann, Z. A. Janowicz, and C. P. Hollenberg. 1995. Stable multicopy integration of vector sequences in Hansenula polymorpha. Appl. Microbiol. Biotechnol. 43: 844-849.
  9. Gerngross, T. U. 2004. Advances in the production of human therapeutic proteins in yeasts and filamentous fungi. Nat. Biotechnol. 22: 1409-1414.
  10. Heo, J. H., W. K. Hong, E. Y. Cho, M. W. Kim, J. Y. Kim, C. H. Kim, S. K. Rhee, and H. A. Kang. 2003. Properties of the Hansenula polymorpha-derived constitutive GAP promoter, assessed using an HSA reporter gene. FEMS Yeast Res. 4: 175-184.
  11. Hollenberg, C. P. and G. Gellissen. 1997. Production of recombinant proteins by methylotrophic yeasts. Curr. Opin. Biotechnol. 8: 554-560.
  12. Hou, J., K. E. J. Tyo, Z. Liu, D. Petranovic, and J. Nielsen. 2012. Metabolic engineering of recombinant protein secretion by Saccharomyces cerevisiae. FEMS Yeast Res. 12: 491-510.
  13. Hovland, P., J. Flick, M. Johnston, and R. A. Sclafani. 1989. Galactose as a gratuitous inducer of GAL gene expression in yeasts growing on glucose. Gene 83: 57-64.
  14. Idiris, A., H. Tohda, H. Kumagai, and K. Takegawa. 2010. Engineering of protein secretion in yeast: strategies and impact on protein production. Appl. Microbiol. Biotechnol. 86: 403-
  15. Jana, S. and J. K. Deb. 2005. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl. Microbiol. Biotechnol. 67: 289-298.
  16. Kang, H. A., E. S. Choi, W. K. Hong, J. Y. Kim, S. M. Ko, J. H. Sohn, and S. K. Rhee. 2000. Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 53: 575-582.
  17. Kang, H. A., J. H. Sohn, M. O. Agaphonov, E. S. Choi, M. D. Ter- Avanesyan, and S. K. Rhee. 2002. Development of expression systems for the production of recombinant proteins in Hansenula polymorpha DL-1. Wiley-VCH: 124-146.
  18. Kang, H. A., W. Kang, W. K. Hong, M. W. Kim, J. Y. Kim, J. H. Sohn, E. S. Choi, K. B. Choe, and S. K. Rhee. 2001. Development of expression systems for the production of recombinant human serum albumin using the MOX promoter in Hansenula polymorpha DL-1. Biotechnol. Bioeng. 76: 175-185.
  19. Kang W. K., E. K. Park, H. S. Lee, B. Y. Park, J. Y. Chang, M. Y. Kim, H. A. Kang, and J. Y. Kim. 2007. A biologically active angiogenesis inhibitor, human serum albumin-TIMP-2 fusion protein, secreted from Saccharomyces cerevisiae. Protein Expr. Purif. 53: 331-338.
  20. Li, W., X. Zhou, and P. Lu. 2004. Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res. Microbiol. 155: 605-606.
  21. Lim, C. Y. and D. H. Yi. 2001. Purification and characterization of recombinant human albumin from Hansenula polymorpha DL-1. Appl. Microbiol. Biotechnol. 29: 248-252.
  22. Oshima, Y. 1982. Regulatory circuits for gene expression: the metabolism of galactose and phosphate. 159-180, The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor. New York.
  23. Parekh, R. N. and K. D. Wittrup. 1997. Expression level tuning for optimal heterologous protein secretion in Saccharomyces cerevisiae. Biotechnol. Prog. 13: 117-122.
  24. Ramos, J., K. Szkutnicka, and V. P. Cirillo. 1989. Characteristics of galactose transport in Saccharomyces cerevisiae cells and reconstituted lipid vesicles. J. Bacteriol. 171: 3539-3544.
  25. Romanos, M. A., C. A. Scorer, and J. J. Clare. 1992. Foreign gene expression in yeast: a review. Yeast 8: 423-488.
  26. Sleep, D., G. P. Belfield, and A. R. Goodey. 1990. The secretion of human serum albumin from the yeast Saccharomyces cerevisiae using five different leader sequences. Biotechnology (NY) 8: 42-46.
  27. Sohn, M. J., D.-B. Oh, E. J. Kim, S. A. Cheon, O. Kwon, J.-Y. Kim, S. Y. Lee, and H. A. Kang. 2012. HpYPS1 and HpYPS7 encode functional aspartyl proteases localized at the cell surface in the thermotolerant methylotrophic yeast Hansenula polymorpha. Yeast 29: 1-16.
  28. Sugio, S., A. Kashima, S. Mochizuki, M. Noda, and K. Kobayashi. 1999. Crystal structure of human serum albumin at 2.5 A resolution. Protein Eng. 12: 439-446.
  29. Vai, M. and L. Brambilla. 2000. Improved secretion of native human insulin-like growth factor 1 from gas1 mutant Saccharomyces cerevisiae cells. Appl. Environ. Microbiol. 66: 5477- 5479.
  30. Youn, J. K., L. Shang, M. I. Kim, C. M. Jeong, H. N. Chang, M. S. Hahm, S. K. Rhee, and H. A. Kang. 2010. Enhanced production of human serum albumin by fed-batch culture of Hansenula polymorpha with high-purity oxygen. J. Microbiol. Biotechnol. 20: 1534-1538.