Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris

Abstract

Human α-defensin 5 (HD5), a small cysteine-rich peptide expressed predominantly in small intestine and female reproductive tissues, plays an important role in innate and adaptive immunity. It is a worthy yet challenging work to produce bioactive recombinant HD5 through the use of bioengineering techniques. Here, we present the expression and purification of recombinant HD5 mature peptide (rmHD5) in Pichia pastoris. To avoid generating unfavorable extra N-terminal amino acids, Red/ET homologous recombination was applied to construct the expression vector pPIC9K-mHD5 by insertion of a polymerase chain reaction-amplified DNA fragment coding for mHD5 into the plasmid pPIC9K, at a position right after the cleavage sequence of Kex2. The pPIC9K-mHD5 vector was transformed into P. pastoris GS115 cells, and positive colonies harboring genomic integration of the multicopy mHD5 nucleotide sequence were screened out and used for fermentation. After high-cell density fermentation of P. pastoris GS115-HD5, a two-step purification strategy of macroporous resin adsorption chromatography followed by cation exchange chromatography was performed to obtain purified rmHD5. The results showed that about 165.0 mg/l of rmHD5 with its intact N-terminal amino acid sequence as revealed by mass spectrometry analysis and amino acid sequencing was produced under optimal bioreactor-culture conditions and that approximately 50% of the initial rmHD5 was recovered after purification. The in vitro experiments revealed that rmHD5 exhibited a prominent antibacterial activity and potency to block human papillomavirus infection. This is the first report on the production and purification of bioactive rmHD5 in P. pastoris. This study also provides considerations for production of other antimicrobial peptides using the P. pastoris expression system.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig.5
Fig. 6

References

  1. 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

  2. Buck CB, Day PM, Thompson CD, Lubkowski J, Lu W, Lowy DR, Schiller JT (2006) Human alpha-defensins block papillomavirus infection. Proc Natl Acad Sci USA 103:1516–1521

  3. Cabral KM, Almeida MS, Valente AP, Almeida FC, Kurtenbach E (2003) Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease. Protein Expr Purif 31:115–122

  4. Chen H, Xu Z, Peng L, Fang X, Yin X, Xu N, Cen P (2006) Recent advances in the research and development of human defensins. Peptides 27:931–940

  5. Cunliffe RN, Rose FR, Keyte J, Abberley L, Chan WC, Mahida YR (2001) Human defensin 5 is stored in precursor form in normal Paneth cells and is expressed by some villous epithelial cells and by metaplastic Paneth cells in the colon in inflammatory bowel disease. Gut 48:176–185

  6. Ericksen B, Wu Z, Lu W, Lehrer RI (2005) Antibacterial activity and specificity of the six human alpha-defensins. Antimicrob Agents Chemother 49:269–275

  7. Frye M, Bargon J, Dauletbaev N, Weber A, Wagner TO, Gropp R (2000) Expression of human alpha-defensin 5 (HD5) mRNA in nasal and bronchial epithelial cells. J Clin Pathol 53:770–773

  8. Ganz T (2002) Immunology. Versatile defensins. Science 298:977–979

  9. Ghosh D, Porter E, Shen B, Lee SK, Wilk D, Drazba J, Yadav SP, Crabb JW, Ganz T, Bevins CL (2002) Paneth cell trypsin is the processing enzyme for human defensin-5. Nat Immunol 3:583–590

  10. Hancock RE, Sahl HG (2006) Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24:1551–1557

  11. Huang H, Yang P, Luo H, Tang H, Shao N, Yuan T, Wang Y, Bai Y, Yao B (2008) High-level expression of a truncated 1,3–1,4-beta-d-glucanase from Fibrobacter succinogenes in Pichia pastoris by optimization of codons and fermentation. Appl Microbiol Biotechnol 78:95–103

  12. Jin F, Xu X, Zhang W, Gu D (2006) Expression and characterization of a housefly cecropin gene in the methylotrophic yeast, Pichia pastoris. Protein Expr Purif 49:39–46

  13. Jones DE, Bevins CL (1992) Paneth cells of the human small intestine express an antimicrobial peptide gene. J Biol Chem 267:23216–23225

  14. Kim JM, Jang SA, Yu BJ, Sung BH, Cho JH, Kim SC (2008) High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage. Appl Microbiol Biotechnol 78:123–130

  15. Leeuw E, Burks SR, Li X, Kao JP, Lu W (2007) Structure-dependent functional properties of human defensin 5. FEBS Lett 581:515–520

  16. Lehrer RI, Ganz T (2002) Defensins of vertebrate animals. Curr Opin Immunol 14:96–102

  17. Li L, Wang JX, Zhao XF, Kang CJ, Liu N, Xiang JH, Li FH, Sueda S, Kondo H (2005) High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris. Protein Expr Purif 39:144–151

  18. Muyrers JP, Zhang Y, Benes V, Testa G, Ansorge W, Stewart AF (2000) Point mutation of bacterial artificial chromosomes by ET recombination. EMBO Rep 1:239–243

  19. Ouellette AJ (2006) Paneth cell alpha-defensin synthesis and function. Curr Top Microbiol Immunol 306:1–25

  20. Plantz BA, Sinha J, Villarete L, Nickerson KW, Schlegel VL (2006) Pichia pastoris fermentation optimization: energy state and testing a growth-associated model. Appl Microbiol Biotechnol 72:297–305

  21. Salzman NH, Ghosh D, Huttner KM, Paterson Y, Bevins CL (2003) Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin. Nature 422:522–526

  22. Svinarich DM, Wolf NA, Gomez R, Gonik B, Romero R (1997) Detection of human defensin 5 in reproductive tissues. Am J Obstet Gynecol 176:470–475

  23. Szyk A, Wu Z, Tucker K, Yang D, Lu W, Lubkowski J (2006) Crystal structures of human alpha-defensins HNP4, HD5, and HD6. Protein Sci 15:2749–2760

  24. Tian ZG, Teng D, Yang YL, Luo J, Feng XJ, Fan Y, Zhang F, Wang JH (2007) Multimerization and fusion expression of bovine lactoferricin derivative LfcinB15–W4, 10 in Escherichia coli. Appl Microbiol Biotechnol 75:117–124

  25. Wang JP, Zhang YM (2005) The application of Red/ET recombination to high efficient gene-targeting vector construction. Yi Chuan 27:953–958

  26. Wang A, Su Y, Cheng T, Zou Z, Wang J (2008) Soluble expression and purification of human alpha-defensin-5 in Escherichia coli. Sheng Wu Gong Cheng Xue Bao 24:291–296

  27. Wei Q, Kim YS, Seo JH, Jang WS, Lee IH, Cha HJ (2005) Facilitation of expression and purification of an antimicrobial peptide by fusion with baculoviral polyhedrin in Escherichia coli. Appl Environ Microbiol 71:5038–5043

  28. White CE, Kempi NM, Komives EA (1994) Expression of highly disulfide-bonded proteins in Pichia pastoris. Structure 2:1003–1005

  29. Wu Z, Ericksen B, Tucker K, Lubkowski J, Lu W (2004) Synthesis and characterization of human a-defensins 4–6. J. Peptide Res. 64:118–125

  30. Yeaman MR, Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55

  31. Yu P (2007) A new approach to the production of the recombinant SOD protein by methylotrophic Pichia pastoris. Appl Microbiol Biotechnol 74:93–98

  32. Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128

  33. Zhang W, Smith LA, Plantz BA, Schlegel VL, Meagher MM (2002) Design of methanol feed control in Pichia pastoris fermentations based upon a growth model. Biotechnol Prog 18:1392–1399

Download references

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (no. 30771892), Academician Fund of Chongqing City (no. CSTC, 2007AB5022), Special Fund of National Key Laboratory of Trauma, Burn and Combined Injury (no. SKLZZ200821), Natural Science Foundation of Chongqing City (no. CSTC, 2008BB5137), and National “863” High-tech Development Plan of China (no. 2007AA02Z152).

Author information

Correspondence to Yongping Su or Junping Wang.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wang, A., Wang, S., Shen, M. et al. High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris . Appl Microbiol Biotechnol 84, 877–884 (2009). https://doi.org/10.1007/s00253-009-2020-x

Download citation

Keywords

  • Human α defensin 5
  • Pichia pastoris
  • Expression
  • Purification
  • Bioactivity