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.
Takahashi
N
N
Research Institute for Food Science, Kyoto University, Japan.
Orita
T
T
Hirose
M
M
D000595:Amino Acid Sequence; D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D002645:Chickens; D002942:Circular Dichroism; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D010446:Peptide Fragments; D011994:Recombinant Proteins; D010047:Ovalbumin; D010450:Endopeptidases