Expression of tropomyosin from Blattella germanica as a recombinant non-fusion protein in Pichia pastoris and comparison of its IgE reactivity with its native counterpart

https://doi.org/10.1016/j.pep.2004.06.009Get rights and content

Abstract

Tropomyosins derived from invertebrates are well-known pan allergens. However, the allergenicities of recombinant tropomyosins are variable. Here, we undertook to compare the IgE-binding reactivities of native and recombinant German cockroach tropomyosins. Native tropomyosin was purified by ammonium sulfate fractionation, hydroxyapatite column chromatography, and electroelution, and recombinant tropomyosin was expressed in Pichia pastoris. The allergenicities of the native and recombinant tropomyosins were compared by ELISA inhibition analysis. Native German cockroach tropomyosin showed 18% IgE-binding reactivity to German cockroach sensitized sera. Recombinant tropomyosin was produced without fusion protein and its N-terminus was blocked like that of the native counterpart. The IgE-binding reactivity of the recombinant was found to be comparable to that of native tropomyosin over the concentration range 1–1000 ng/ml by ELISA inhibition testing. Recombinant German cockroach tropomyosin expressed in Pichia pastoris showed better allergenicity than that expressed in Escherichia coli. Other factors in addition to the structural differences of native and recombinant proteins may also influence the IgE reactivities of tropomyosins.

Section snippets

Purification of native Bla g 7

Cockroaches (60 g) were homogenized in 200 ml of 6 mM of 2-mercaptoethanol in a Waring blender at 4 °C All subsequent steps were performed at 4 °C unless indicated otherwise. The homogenate was pressed through three layers of cheesecloth and mixed with 20 volumes (4 L) of ethanol. The solution was then allowed to stand for 1 h and centrifuged for 30 min at 3000 rpm. The supernatant was discarded and the sediment was resuspended in ether and air-dried. The dried residue was then extracted with gentle

Purification of native tropomyosin and Pichia-expressed recombinant tropomyosin

Native tropomyosin was purified from the whole body extract of German cockroach by ammonium sulfate fractionation, hydroxyapatite column chromatography, and electroelution. The yield of native allergen was 0.08% (w/w) (Table 1). Recombinant tropomyosin was expressed as a non-fusion protein in P. pastoris and was purified using the same procedure as that used for native tropomyosin purification. The yield of purified recombinant tropomyosin was approximately 7.2 mg/L on yeast culture. We were

Discussion

Various expression systems have been designed to produce recombinant allergens to overcome the clinical limitations of natural allergen extracts, i.e., stability, contamination, and variations in composition and quantity [27], [28], [29], [30]. The methylotrophic yeast P. pastoris is one of several valuable tools, which are capable of performing post-translational modifications, such as, disulfide bond formation, glycosylation, acetylation, and protein folding [31]. For example, the biological

Acknowledgements

We thank Miss Kyung-Eun Lee for assistance with the skin prick test and Professor Kyu-Earn Kim for valuable discussions. This work was supported by Grant No. R01-2002-000-00243-0 from the Basic Research Program of the Korean Science and Engineering Foundation.

References (44)

  • H.S. Nelson et al.

    Studies of allergen extract stability: the effect of dilution and mixing

    J. Allergy Clin. Immunol

    (1996)
  • J.L. Cereghino et al.

    Heterologous protein expression in the methylotrophic yeast Pichia pastoris

    FEMS Microbiol. Rev

    (2000)
  • F. Shakib et al.

    A mite subversive: cleavage of CD23 and CD25 by Der p 1 enhances allergenicity

    Immunol. Today

    (1998)
  • E.A. Best et al.

    A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen

    Protein Expr. Purif

    (2000)
  • D.H. Heeley et al.

    Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle α α-tropomyosin

    J. Biol. Chem

    (1989)
  • A. Otsu et al.

    Genetic and environmental factors of atopy

    Allergol. Intl

    (2002)
  • R.W. Heald et al.

    The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin

    J. Biol. Chem

    (1988)
  • M.L. Patterson et al.

    Characterization and comparison of commercially available German and American cockroach allergen extracts

    Clin. Exp. Allergy

    (2002)
  • R. Hiller et al.

    Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment

    FASEB. J

    (2002)
  • C. Harwanegg et al.

    Microarrayed recombinant allergens for diagnosis of allergy

    Clin. Exp. Allergy

    (2003)
  • A. Pomes et al.

    Can knowledge of the molecular structure of allergens improve immunotherapy

    Curr. Opin. Allergy Clin. Immunol

    (2001)
  • L. Kazemi-Shirazi et al.

    Recombinant marker allergens: diagnostic gatekeepers for the treatment of allergy

    Int. Arch. Allergy Immunol

    (2002)
  • Cited by (14)

    • Auto-induction for high yield expression of recombinant novel isoallergen tropomyosin from King prawn (Melicertus latisulcatus) for improved diagnostics and immunotherapeutics

      2014, Journal of Immunological Methods
      Citation Excerpt :

      rTM yields achieved are published for house dust mites (Aki et al., 1994; Asturias et al., 1998), cockroach (Jeong et al., 2004) and chicken (Hilario et al., 2001). Despite the different sources of TM and expression systems, the purified rTM yields reported are relatively low and vary between 7.2 mg/l (Jeong et al., 2004) and 26 mg/l (Asturias et al., 1998). However, larger quantities are needed as standards for the detection of allergens, diagnostics for allergic sensitization and development of immunotherapeutics.

    • Cockroach allergens Per a 3 are oligomers

      2010, Developmental and Comparative Immunology
    • Mannose addition by yeast Pichia Pastoris on recombinant HER-2 protein inhibits recognition by the monoclonal antibody herceptin

      2009, Vaccine
      Citation Excerpt :

      These facts demonstrate the need for further study of HER-2-associated immunity, and for large-scale production of HER-2 protein, to use as a model for development of vaccine applications. Yeast combines many advantages of prokaryotic recombinant protein production with the post-translational modifications, native folding and antigenic properties evidenced in mammalian cells [12,13]. We, therefore, generated recombinant yeast to express HER-2 protein.

    View all citing articles on Scopus
    View full text