Skip to main content
SpringerLink
Log in

Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The anthrax toxin is an AB-type bacterium toxin composed of the protective antigen (PA) as the cell-binding B component, and the lethal factor (LF) and edema toxin (EF) as the catalytic A components. The PA component is a key factor in anthrax-related research and recombinant PA can be produced in general in Escherichia coli. However, such recombinant PA always forms inclusion bodies in the cytoplasm of E. coli, making difficult the procedure of its purification. In this study, we found that the solubility of recombinant PA was dramatically enhanced by fusion with glutathione S-transferase (GST) and an induction of its expression at 28°C. The PA was purified to high homogeneity and a yield of 3 mg protein was obtained from 1 l culture by an affinity-chromatography approach. Moreover, we expressed and purified three PA mutants, I394C, A396C, and N435C, which were impaired in expression in previous study. Among them, a novel mutant N435C which conferred dominant-negative inhibitory activity on PA was identified. This new mutant may be useful in designing new antitoxin for anthrax prophylaxis and therapy.

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

  • Ahuja N, Kumar P, Bhatnagar R (2001) Rapid purification of recombinant anthrax-protective antigen under nondenaturing conditions. Biochem Biophys Res Commun 286:6–11

    Article  CAS  Google Scholar 

  • Ahuja N, Kumar P, Alam S, Gupta M, Bhatnagar R (2003) Deletion mutants of protective antigen that inhibit anthrax toxin both in vitro and in vivo. Biochem Biophys Res Commun 307:446–450

    Article  CAS  Google Scholar 

  • Baillie LW (2006) Past, imminent and future human medical countermeasures for anthrax. J Appl Microbiol 101:594–606

    Article  CAS  Google Scholar 

  • Barth H, Aktories K, Popoff MR, Stiles BG (2004) Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins. Microbiol Mol Biol Rev 68:373–402

    Article  CAS  Google Scholar 

  • Benson E, Huynh P, Finkelstein A, Collier R (1998) Identification of residues lining the anthrax protective antigen channel. Biochemistry 37:3941–3948

    Article  CAS  Google Scholar 

  • Bradley KA, Mogridge J, Mourez M, Collier RJ, Young JA (2001) Identification of the cellular receptor for anthrax toxin. Nature 414:225–229

    Article  CAS  Google Scholar 

  • Cao S, Liu Z, Guo A, Li Y, Zhang C, Gaobing W, Chunfang F, Tan Y, Chen H (2008) Efficient production and characterization of Bacillus anthracis lethal factor and a novel inactive mutant rLFm-Y236F. Protein Expr Purif 59:25–30

    Article  CAS  Google Scholar 

  • Chang H, Tsai M, Chung C, Chen P, Hu H, Kau J, Huang H, Lin H, Sun D (2006) Single-step purification of recombinant anthrax lethal factor from periplasm of Escherichia coli. J Biotechnol 126:277–285

    Article  CAS  Google Scholar 

  • Chauhan V, Singh A, Waheed SM, Singh S, Bhatnagar R (2001) Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli. Biochem Biophys Res Commun 283:308–315

    Article  CAS  Google Scholar 

  • Coulson NM, Fulop M, Titball RW (1994) Bacillus anthracis protective antigen, expressed in Salmonella typhimurium SL 3261, affords protection against anthrax spore challenge. Vaccine 12:1395–1401

    Article  CAS  Google Scholar 

  • Cunningham K, Lacy DB, Mogridge J, Collier RJ (2002) Mapping the lethal factor and edema factor binding sites on oligomeric anthrax protective antigen. Proc Natl Acad Sci U S A 99:7049–7053

    Article  CAS  Google Scholar 

  • Dian C, Eshaghi S, Urbig T, McSweeney S, Heijbel A, Salbert G, Birse D (2002) Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins. J Chromatogr B Analyt Technol Biomed Life Sci 769:133–144

    Article  CAS  Google Scholar 

  • Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, Ahn NG, Oskarsson MK, Fukasawa K, Paull KD, Vande Woude GF (1998) Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280:734–737

    Article  CAS  Google Scholar 

  • Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotech 17:353–358

    Article  CAS  Google Scholar 

  • Gupta P, Waheed SM, Bhatnagar R (1999) Expression and purification of the recombinant protective antigen of Bacillus anthracis. Protein Expr Purif 16:369–376

    Article  CAS  Google Scholar 

  • Hammarstrom M, Hellgren N, van Den Berg S, Berglund H, Hard T (2002) Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli. Protein Sci 11:313–321

    Article  CAS  Google Scholar 

  • Janowiak B, Finkelstein A, Collier R (2009) An approach to characterizing single-subunit mutations in multimeric prepores and pores of anthrax protective antigen. Protein Science 18:348–358

    Article  CAS  Google Scholar 

  • Klimpel KR, Molloy SS, Thomas G, Leppla SH (1992) Anthrax toxin protective antigen is activated by a cell surface protease with the sequence specificity and catalytic properties of furin. Proc Natl Acad Sci U S A 89:10277–10281

    Article  CAS  Google Scholar 

  • Laird MW, Zukauskas D, Johnson K, Sampey GC, Olsen H, Garcia A, Karwoski JD, Cooksey BA, Choi GH, Askins J, Tsai A, Pierre J, Gwinn W (2004) Production and purification of Bacillus anthracis protective antigen from Escherichia coli. Protein Expr Purif 38:145–152

    Article  CAS  Google Scholar 

  • Leppla SH (1982) Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells. Proc Natl Acad Sci U S A 79:3162–3166

    Article  CAS  Google Scholar 

  • Lu J, Wei D, Wang Y, Wang G (2009) High-level expression and single-step purification of recombinant Bacillus anthracis protective antigen from Escherichia coli. Biotechnol Appl Bioc 52:107–112

    Article  CAS  Google Scholar 

  • Miller J, McBride BW, Manchee RJ, Moore P, Baillie LW (1998) Production and purification of recombinant protective antigen and protective efficacy against Bacillus anthracis. Lett Appl Microbiol 26:56–60

    Article  CAS  Google Scholar 

  • Mogridge J, Mourez M, Collier RJ (2001) Involvement of domain 3 in oligomerization by the protective antigen moiety of anthrax toxin. J Bacteriol 183:2111–2116

    Article  CAS  Google Scholar 

  • Mogridge J, Cunningham K, Lacy DB, Mourez M, Collier RJ (2002) The lethal and edema factors of anthrax toxin bind only to oligomeric forms of the protective antigen. Proc Natl Acad Sci U S A 99:7045–7048

    Article  CAS  Google Scholar 

  • Mourez M, Yan M, Lacy DB, Dillon L, Bentsen L, Marpoe A, Maurin C, Hotze E, Wigelsworth D, Pimental RA, Ballard JD, Collier RJ, Tweten RK (2003) Mapping dominant-negative mutations of anthrax protective antigen by scanning mutagenesis. Proc Natl Acad Sci U S A 100:13803–13808

    Article  CAS  Google Scholar 

  • Petosa C, Collier RJ, Klimpel KR, Leppla SH, Liddington RC (1997) Crystal structure of the anthrax toxin protective antigen. Nature 385:833–838

    Article  CAS  Google Scholar 

  • Qa'dan M, Christensen KA, Zhang L, Roberts TM, Collier RJ (2005) Membrane insertion by anthrax protective antigen in cultured cells. Mol Cell Biol 25:5492–5498

    Article  Google Scholar 

  • Rainey GJ, Young JA (2004) Antitoxins: novel strategies to target agents of bioterrorism. Nat Rev Microbiol 2:721–726

    Article  Google Scholar 

  • Rhie GE, Park YM, Han JS, Yu JY, Seong WK, Oh HB (2005) Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis. FEMS Immunol Med Microbiol 45:341–347

    Article  CAS  Google Scholar 

  • Scobie HM, Rainey GJ, Bradley KA, Young JA (2003) Human capillary morphogenesis protein 2 functions as an anthrax toxin receptor. Proc Natl Acad Sci U S A 100:5170–5174

    Article  CAS  Google Scholar 

  • Sellman BR, Mourez M, Collier RJ (2001) Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax. Science 292:695–697

    Article  CAS  Google Scholar 

  • Sharma M, Swain PK, Chopra AP, Chaudhary VK, Singh Y (1996) Expression and purification of anthrax toxin protective antigen from Escherichia coli. Protein Expr Purif 7:33–38

    Article  CAS  Google Scholar 

  • Sorensen HP, Mortensen KK (2005) Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 4:1

    Article  Google Scholar 

  • Sun J, Lang AE, Aktories K, Collier RJ (2008) Phenylalanine-427 of anthrax protective antigen functions in both pore formation and protein translocation. Proc Natl Acad Sci U S A 105:4346–4351

    Article  CAS  Google Scholar 

  • Yan M, Collier RJ (2003) Characterization of dominant-negative forms of anthrax protective antigen. Mol Med 9:46–51

    CAS  Google Scholar 

  • Yan M, Roehrl MH, Basar E, Wang JY (2008) Selection and evaluation of the immunogenicity of protective antigen mutants as anthrax vaccine candidates. Vaccine 26:947–955

    Article  CAS  Google Scholar 

  • Young JA, Collier RJ (2007) Anthrax toxin: receptor binding, internalization, pore formation, and translocation. Annu Rev Biochem 76:243–265

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Key Project of Chinese Ministry of Education (No. 306013) and China National Basic Research (973) Program (No.2006CB504401).

Author information

Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Gaobing Wu, Chunfang Feng, Aizhen Guo, Sha Cao, Junli Dong, Ling Lin & Ziduo Liu

  2. College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Yuzhi Hong

Authors
  1. Gaobing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chunfang Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuzhi Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Aizhen Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sha Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Junli Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ling Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ziduo Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ziduo Liu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wu, G., Feng, C., Hong, Y. et al. Soluble expression and purification of the anthrax protective antigen in E. coli and identification of a novel dominant-negative mutant N435C. Appl Microbiol Biotechnol 87, 609–616 (2010). https://doi.org/10.1007/s00253-010-2495-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2495-5

Keywords

Search

Navigation