Publications by authors named "Astrid W van Leeuwen"

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Characterization of natural Dac g 1 variants: an alternative to recombinant group 1 allergens.

J Allergy Clin Immunol 2004 Nov;114(5):1124-30

Department of Immunopathology, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.

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 (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr 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 LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr 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.
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November 2004