Production of chicken ovalbumin in Escherichia coli.

For better understanding of the structure-function relationship in serine proteinase inhibitors, a protein engineering approach for converting non-inhibitory chicken ovalbumin (Ova) to the inhibitory form would be a highly useful model system. A prerequisite expression system for the Ova-encoding gene (Ova) was established in this study. The Ova gene was expressed in Escherichia coli with high yield using the T7 phage promoter; the amount of the recombinant Ova (re-Ova) was 29.4% of cellular proteins. SDS-PAGE and Western blotting analysis revealed that re-Ova immunoreacting with the egg ovalbumin antibody is not glycosylated. The re-Ova was purified by anion exchange chromatography into homogeneity, as evaluated by SDS-PAGE. Amino-acid and N-terminal sequence analyses confirmed that the purified product had the correct sequence designed for Ova production. As for secondary structure, re-Ova showed a far-UV circular dichroism spectrum indistinguishable from natural egg Ova. Furthermore, the proteolytic fragmentation pattern that should reflect protein conformation was exactly the same for the natural egg and re-Ova. Using the proteolytic fragments, the identity of the internal sequences for the natural and re- proteins was confirmed.