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High-Level Expression of Peanut Allergens Affected by Rare Codon Usage

37C). Of the total E. coli lysates, 1 ml each was harvested after 2, 4, and 20 hours (overnight incubation) by centrifugation, then resuspended in 100 l of reducing sample buffer. The samples were analyzed on SDS-15% PAGE and visualized by staining with Coomassie Brilliant Blue stain. Proteins were transferred to a nitrocellulose membrane for Western blot experiments. A serum of a peanut allergen was diluted 1:50 and used as the first antibody. The specific binding of the IgE to the recombinant Ara h 2 was detected by incubating the membrane with alkaline-phophatase-conjugated antihuman IgE as the second antibody, and bands were visualized with nitroblue tetrazolium chloride including 5-bromo-4-chloro-3-indolyl phosphate potassium salt.

REFERENCES

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  3. Schenk, P.M., et al. (1995) Biotechniques 13: 14-18.

  4. Kleber-Janke, T., et al. (1999) Int. Arch. Allergy Immunol. 119: 265-274.

  5. Burks, A.W., et al. (1995) J. Clin. Invest. 96: 1715-1721.

  6. Carstens C.-P. and Waesche A. (1999) Strategies 12: 49-51.

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