Characterization of natural Dac g 1 variants: an alternative to recombinant group 1 allergens
| Autor: | Pleuni G. de Heer, Astrid van Leeuwen, Rob C. Aalberse, Miranda Dieker, Erica van Oort, Ronald van Ree |
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| Přispěvatelé: | Landsteiner Laboratory, Experimental Immunology |
| Rok vydání: | 2004 |
| Předmět: |
Glycosylation
Immunology Size-exclusion chromatography law.invention Pichia pastoris Affinity chromatography law Immunology and Allergy Humans Amino Acid Sequence Cloning Molecular Plant Proteins chemistry.chemical_classification biology Molecular mass Chemistry Hydrophilic interaction chromatography Allergens Antigens Plant biology.organism_classification In vitro Recombinant Proteins Amino acid Biochemistry Recombinant DNA |
| Zdroj: | Journal of allergy and clinical immunology, 114(5), 1124-1130. Mosby Inc. |
| ISSN: | 0091-6749 |
| Popis: | Background Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. Objective Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. Methods Dac g 1 was cloned and expressed in the yeast Pichia pastoris . Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. Results Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (M r ) on SDS-PAGE of 33 kd (HM r ), 30 kd (IM r ) and 28 kd (LM r ). Extraction of IM r Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HM r and IM r Dac g 1 differs at 2 positions, and LM r Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LM r molecule. IM r Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HM r Dac g 1. Conclusions Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens. |
| Databáze: | OpenAIRE |
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