Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage.
We cloned two distinct cDNAs for enteropeptidase (EP) from the intestine of the medaka, Oryzias latipes, which is a small freshwater teleost. The mRNAs code for EP-1 (1,036 residues) and EP-2 (1,043 residues), both of which have a unique, conserved domain structure of the N-terminal heavy chain and C-terminal catalytic serine protease light chain. When compared with mammalian EP serine proteases, the medaka enzyme exhibited extremely low amidolytic activity for small synthetic peptide substrates. Twelve mutated forms of the medaka EP protease were produced by site-directed mutagenesis. Among them, one mutant protease, E173A, was found to have considerably reduced nonspecific hydrolytic activities both for synthetic and protein substrates without serious reduction of its Asp-Asp-Asp-Asp-Lys (D(4)K)-cleavage activity. For the cleavage of fusion proteins containing a D(4)K-cleavage site, the medaka EP proteases were shown to have advantages over their mammalian counterparts. Based on our present data, we propose that the E173A mutant is the most appropriate protease to specifically cleave proteins containing the D(4)K cleavage sequence.
Ogiwara
Katsueki
K
Laboratory of Molecular and Cellular Interactions, Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810, Japan.
Takahashi
Takayuki
T
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008322:Mammals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009990:Oryzias / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D018076:DNA, Complementary; D050505:Mutant Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases