Skip to main content

Advertisement

SpringerLink
Log in

High efficiency preparation of bioactive human α-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression

  • Methods and Protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Human α-defensin 6 (HD6), a small cysteine-rich cationic peptide specially expressed in epithelial cells of digestive tract, may play a crucial role in mucosal immunity. This is the first report on efficient production of bioactive HD6 through a gene-engineering approach in Escherichia coli. The recombinant plasmid pET32a-omHD6 was primarily constructed by inserting a PCR fragment encoding mature HD6 peptide (mHD6) preceded by an enterokinase recognition sequence into the expression vector pET32a(+), in frame with the upstream thioredoxin (TrxA) gene. Under optimized expression conditions, a high percentage (>60%) of soluble TrxA-omHD6 fusion protein was obtained with a yield of about 1.69 g/l, and the theoretical productivity of recombinant mHD6 (rmHD6) reached 0.38 g/l. A feasible three-step purification strategy involving nickel-sepharose chromatography, enterokinase-cleavage and cation exchange chromatography was developed to purify rmHD6, followed by characteristic identifications by Western blot, mass spectrometry and sequencing. About 102 mg/l of rmHD6 with its intact N-terminal amino acid sequence was finally achieved. The in vitro experiments showed that rmHD6 possesses high potency to inhibit herpes simplex virus-2 infection. This work settles substantial foundation for further functional study of HD6.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

References

  • Ario de Marco (2009) Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb Cell Factories 26:1–18

    Google Scholar 

  • 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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Cipakova I, Gasperik J, Hostinova E (2006) Expression and purification of human antimicrobial peptide, dermcidin, in Escherichia coli. Protein Expr Purif 45:269–274

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Hazrati E, Galen B, Lu W, Wang W, Ouyang Y, Keller MJ, Lehrer RI, Herold BC (2006) Human α- and β-defensins block multiple steps in herpes simplex virus infection. J Immunol 177:8658–8666

    CAS  Google Scholar 

  • Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Jones DE, Bevins CL (1993) Defensin 6 mRNA in human Paneth cells: implications for antimicrobial peptides in host defense of the human bowel. FEBS Lett 315:187–192

    Article  CAS  Google Scholar 

  • Lee JH, Minn I, Park CB, Kim SC (1998) Acidic peptide-mediated expression of the antimicrobial peptide buforin II as tandem repeats in Escherichia coli. Protein Expr Purif 12:53–60

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Nam MJ, Kee MK, Kuick R, Hanash SM (2005) Identification of α-defensin 6 as a potential biomarker in colon adenocarcinoma. J Biol Chem 280:8260–8265

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Peng L, Xu Z, Fang X, Wang F, Yang S, Cen P (2004) Preferential codons enhancing the expression level of human β-defensin-2 in recombinant Escherichia coli. Protein Pept Lett 11:339–344

    Article  CAS  Google Scholar 

  • Porter EM, van Dam E, Valore EV, Ganz T (1997) Broad-spectrum antimicrobial activity of human intestinal defensin 5. Infect Immun 65:2396–2401

    CAS  Google Scholar 

  • Porter EM, Poles MA, Lee JS, Naitoh J, Bevins CL, Ganz T (1998) Isolation of human intestinal defensins from ileal neobladder urine. FEBS Lett 434:272–276

    Article  CAS  Google Scholar 

  • Prinz WA, Aslund F, Holmgren A, Beckwith J (1997) The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm. J Biol Chem 272:15661–15667

    Article  CAS  Google Scholar 

  • Rao XC, Li S, Hu JC, Jin XL, Hu XM, Huang JJ, Chen ZJ, Zhu JM, Hu FQ (2004) A novel carrier molecule for high-level expression of peptide antibiotics in Escherichia coli. Protein Expr Purif 36:11–18

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Tian ZG, Dong TT, Yang YL, Teng D, Wang JH (2009) Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3). Appl Microbiol Biotechnol 83:143–149

    Article  CAS  Google Scholar 

  • Wang A, Wang S, Shen M, Chen F, Zou Z, Ran X, Cheng T, Su Y, Wang J (2009) High level expression and purification of bioactive human α-defensin 5 mature peptide in Pichia pastoris. Appl Microbiol Biotechnol 84:877–884

    Article  CAS  Google Scholar 

  • Wehkamp J, Chu H, Shen B, Feathers RW, Kays RJ, Lee SK, Bevins CL (2006) Paneth cell antimicrobial peptides: topographical distribution and quantification in human gastrointestinal tissues. FEBS Lett 580:5344–5350

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Xu X, Jin F, Yu X, Ji S, Wang J, Cheng H, Wang C, Zhang W (2007) Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr Purif 53:293–301

    Article  CAS  Google Scholar 

  • Zhong Z, Xu Z, Peng L, Huang L, Fang X, Cen P (2006) Tandem repeat mhBD2 gene enhance the soluble fusion expression of hBD2 in Escherichia coli. Appl Microbiol Biotechnol 71:661–667

    Article  CAS  Google Scholar 

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), and National “863” High-tech Development Plan of China (2007AA02Z152).

Author information

Authors and Affiliations

  1. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury of PLA, Third Military Medical University, Chongqing, 400038, People’s Republic of China

    Aiping Wang, Yongping Su, Song Wang, Mingqiang Shen, Fang Chen, Mo Chen, Xinze Ran, Tianmin Cheng & Junping Wang

Authors
  1. Aiping Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yongping Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Song Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mingqiang Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mo Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xinze Ran
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tianmin Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Junping Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Junping Wang.

Elecronic Supplementary Materials

Below is the link to the electronic supplementary material.

Fig. S1

Purified rmHD6 analyzed by reversed phase high performance liquid chromatography. The purity of rmHD6 is 74.7% (GIF 13 kb)

Fig. S2

Purified rmHD6 and Dithiothreitol-reduced rmHD6 analyzed by MALDI-TOF mass spectrometry. Molecular mass of rmHD6 is 3,707.6 Da (a), and reduced rmHD6 is 3713.4 Da (b) (GIF 34 kb)

High resolution image (TIFF 7756 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, A., Su, Y., Wang, S. et al. High efficiency preparation of bioactive human α-defensin 6 in Escherichia coli Origami(DE3)pLysS by soluble fusion expression. Appl Microbiol Biotechnol 87, 1935–1942 (2010). https://doi.org/10.1007/s00253-010-2688-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2688-y

Keywords

Search

Navigation