Formation of covalent hybrids from amino-terminal and carboxy-terminal fragments of two ovomucoid third domains.
Turkey ovomucoid third domain and Gambel's quail ovomucoid third domain were converted to their modified forms by specific hydrolysis of the Leu18-Glu19 reactive site peptide bond. The modified inhibitors were reduced, yielding in each case two chains, the NH2-terminal 1-18 and COOH-terminal 19-56, which could be separated by gel exclusion chromatography under reducing conditions. The chains were then converted to mixed disulfides with glutathione. The NH2-terminal peptide of turkey domain was mixed with COOH peptide of Gambel's quail domain and allowed to form interchain disulfide bridges to produce "Turbel" hybrid. The hybrid was obtained in 20% yield and was active as a proteinase inhibitor. This modified hybrid could be purified to virtual homogeneity by ion-exchange chromatography. The reactive site peptide bond was then enzymatically synthesized yielding virgin "Turbel" hybrid. The amino acid sequence of this hybrid was in exact accord with expectations. We have shown further that the reactive site hydrolysis, disulfide reduction, reoxidation, and reactive site bond resynthesis cycle do not affect Kassoc for chymotrypsin. Thus, if the results described here are general, we have a useful method for generating many avian ovomucoid third domain variants.
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002918:Chymotrypsin; D004527:Egg Proteins; D010059:Ovomucin; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D055503:Protein Multimerization; D014422:Turkeys
D004527:Egg Proteins; D010446:Peptide Fragments; D010059:Ovomucin; D002918:Chymotrypsin