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
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)
- et al.
Allergen standardization
J. Allergy Clin. Immunol
(1991) - et al.
Allergen immunotherapy: current and new therapeutic strategies
Allergol. Intl
(2002) - et al.
Immunochemical characterization of recombinant and native tropomyosin as a new allergen from the house dust mite, Dermatophagoides farinae
J. Allergy Clin. Immunol
(1995) - et al.
Cockroach allergens and asthma in Brazil: identification of tropomyosin as a major allergen with potential cross reactivity with mite and shrimp allergens
J. Allergy Clin. Immunol
(1999) - et al.
Sequencing and high level of expression in Escherichia coli of the tropomyosin allergen (Der p 10) from Dermatophagoides pteronyssinus
Biochem. Biophys. Acta
(1998) - et al.
The amino terminus of muscle tropomyosin is a major determinant for function
J. Biol. Chem
(1990) - et al.
High-level production of functional muscle α-tropomyosin in Pichia pastoris
Biochem. Biophys. Res. Commun
(2001) - et al.
Conditions affecting production of functional muscle recombinant α-tropomyosin in Saccharomyces cerevisiae
Protein Expr. Purif
(2003) Preparation and identification of α and β tropomyosins
Methods Ezymol
(1982)- et al.
False-positive skin prick test responses to commercially available dog dander extracts caused by contamination with house dust mite (Dermatophagoides pteronyssinus) allergens
J. Allergy Clin. Immunol
(1996)
Studies of allergen extract stability: the effect of dilution and mixing
J. Allergy Clin. Immunol
Heterologous protein expression in the methylotrophic yeast Pichia pastoris
FEMS Microbiol. Rev
A mite subversive: cleavage of CD23 and CD25 by Der p 1 enhances allergenicity
Immunol. Today
A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen
Protein Expr. Purif
Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle α α-tropomyosin
J. Biol. Chem
Genetic and environmental factors of atopy
Allergol. Intl
The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin
J. Biol. Chem
Characterization and comparison of commercially available German and American cockroach allergen extracts
Clin. Exp. Allergy
Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment
FASEB. J
Microarrayed recombinant allergens for diagnosis of allergy
Clin. Exp. Allergy
Can knowledge of the molecular structure of allergens improve immunotherapy
Curr. Opin. Allergy Clin. Immunol
Recombinant marker allergens: diagnostic gatekeepers for the treatment of allergy
Int. Arch. Allergy Immunol
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 MethodsCitation 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 ImmunologyMannose addition by yeast Pichia Pastoris on recombinant HER-2 protein inhibits recognition by the monoclonal antibody herceptin
2009, VaccineCitation 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.
Non-fusion and fusion expression of β-galactosidase from Lactobacillus bulgaricus in Lactococcus lactis
2008, Biomedical and Environmental SciencesCharacterization of Hum j 6, a Major Allergen From Humulus japonicus Pollen, the Primary Cause of Weed Pollinosis in East Asia
2023, Allergy, Asthma and Immunology Research