Cross-reactivity and improved digestibility of maize γ-zein

Sung Ho Lee, Purdue University


Cross-reactivity of antibodies against almond major protein (AMP) with cereal proteins may cause problems in detecting almonds in cereal products when antibody-based assays are used for such detection. Rabbit polyclonal IgG produced against AMP were used to test cross-reactivity with protein extracts from maize and other cereal grains. Gradient SDS-PAGE followed by Western blotting was performed, and comparably high cross-reactivity was chemiluminescently detected in maize. The anti-AMP IgG affinity chromatographically-purified maize proteins were electrophoresed on a 10% monomer acrylamide gel, followed by immunoblotting with rabbit anti-50 kDa γ-zein antiserum. Results indicated that rabbit anti-AMP antiserum cross-reacted with the 50 kDa maize γ-zein protein and, to a lesser extent, the 27kDa γ-zein. The 50 kDa maize γ-zein also reacted with anti-AMP IgE from pooled human sera from patients with confirmed almond allergies. Whether or not the cross-reactivity of maize γ-zein against patient IgE is relevant to human allergies in vivo remains to be investigated. In addition, maize γ-zein was studied for its digestibility. γ-Zein is a class of maize zein storage proteins that, due to their localization at the protein body periphery, are critical to digestibility characteristics of all zeins. Improvement of maize (and sorghum) protein digestibility has been linked to the digestibility of the γ-zein protein (sorghum equivalent, γ-kafirin). For this reason, the 27 kDa major γ-zein was modified to improve its digestibility through site-directed mutagenesis. A 27 kDa γ-zein cDNA was subcloned into pGEM-11Zf(+) to undergo mutagenesis at 155th Cys to Ala and 181st Gln to Arg. The resulting mutant proteins were expressed in E. coli using the pET24-(a) expression vector under IPTG induction for 3 hours. The newly introduced Ala and Arg residues were expected to play a role in making the protein more accessible to protease leading to increased digestibility. Using the pET24 vector, the Cys155Ala mutant protein was dramatically more digestible to pepsin and trypsin, and the Gln181Arg mutant protein was slightly more soluble in 20 mM Tris-HCl, pH 8.5 compared to the wild-type and Cys155Ala mutant. Elimination of one internal disulfide bond resulted in a highly digestible recombinant protein.




Hamaker, Purdue University.

Subject Area

Food science

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