title: Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue.
abstract: Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.
authors.affiliation: Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kojima
authors.forename: N
authors.initials: N
authors.lastname: Fushimi
authors.forename: A
authors.initials: A
authors.lastname: Ikeda
authors.forename: I
authors.initials: I
authors.lastname: Kumagai
authors.forename: K
authors.initials: K
authors.lastname: Miura
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010059:Ovomucin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D011993:Recombinant Fusion Proteins; D010059:Ovomucin