Expression of recombinant Ara h 6 in Pichia pastoris but not in Escherichia coli preserves allergic effector function and allows assessment of specific mutations.
METHODS
Ara h 6 has recently been recognized as an important peanut allergen. Recombinant allergens have been used for analysis of IgE binding, but have not been used to analyze the allergic effector activity that is more relevant to allergic reactions.


RESULTS
Ara h 6 was expressed as a recombinant protein in both Escherichia coli and Pichia pastoris (rAra h 6-E. coli and rAra h 6-Pichia, respectively). Effector activity was assayed by measuring degranulation of RBL SX-38 cells sensitized with IgE from patients with severe peanut allergy. Compared to native Ara h 6 (nAra h 6), rAra h 6-Pichia had intact effector function whereas rAra h 6-E. coli had significantly reduced function. The lower effector activity in rAra h 6-E. coli compared to nAra h 6 and rAra h 6-Pichia did not appear to be due to differences in posttranslational modifications (analyzed by mass spectrometry and staining for carbohydrates) and may be due to subtle alteration(s) of folding seen on CD analysis and on nonreduced gels. Finally, we introduced point mutations in four important IgE-binding linear epitopes of Ara h 6 and found dramatically reduced allergic effector activity.


CONCLUSIONS
Our studies demonstrate the utility of fully functional rAra h 6-Pichia as a starting point for analysis of specific mutations that adversely affect allergic effector function.
Department of Medicine, Division of Allergy and Clinical Immunology, University of Colorado Denver, Aurora, CO 80045, USA.
Yonghua
Y
Zhuang
Sandy
S
Durrani
Brittany D M
BD
Hodges
Stephen C
SC
Dreskin
Xueni
X
Chen
D055516:2S Albumins, Plant / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000818:Animals; D052179:Antigens, Plant / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015549:Basophil Degranulation Test; D001491:Basophils / Q000276:immunology*; D002460:Cell Line; D002942:Circular Dichroism; D002999:Clone Cells; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D007073:Immunoglobulin E / Q000032:analysis; D050505:Mutant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D021183:Peanut Hypersensitivity / Q000097:blood / Q000276:immunology*; D010843:Pichia / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D055516:2S Albumins, Plant; D052179:Antigens, Plant; C492737:Ara h 6 allergen, Arachis hypogaea; D000939:Epitopes; D050505:Mutant Proteins; D011994:Recombinant Proteins; D007073:Immunoglobulin E