Modified enteropeptidase protein
AssigneeNational University Corporation Hokkaido University
InventorsKatsueki Ogiwara, Takayuki Takahashi
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Abstract
Disclosed are novel enteropeptidase polypeptides, polynucleotides encoding the polypeptides, nucleotide constructs, vectors, host cells comprising the polynucleotides, and methods for producing the polypeptides and polynucleotides. Such polypeptides are useful as protein engineering tool for enzymatic cleavage of fusion proteins. Also provided are kits comprising the polypeptides of the invention.
Claims48
An isolated nucleic acid molecule comprising
- (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:4 or a proteolytically active fragment thereof;
- or
- (b) a nucleic acid molecule encoding a variant of the amino acid sequence of SEQ ID NO:2 wherein the amino acid at an amino acid residue position selected from the group consisting of position 63, position 105, position 144, position 173, and position 193 is mutated, and wherein the amino acid positions are numbered according to the amino acid sequence set forth in SEQ ID NO:65, or a proteolytically active fragment thereof having enteropeptidase activity;
- or
- (c) a nucleic acid molecule which is the complement of (a) or (b).
An isolated enteropeptidase selected from the group consisting of:
- (a) an enteropeptidase comprising the amino acid sequence of SEQ ID NO:4;
- (b) a variant of the amino acid sequence of SEQ ID NO:2 wherein the amino acid at an amino acid residue position selected from the group consisting of position 63, position 105, position 144, position 173, and position 193 is mutated;
- and
- (c) an enteropeptidase comprising a proteolytically active fragment of the amino acid sequence of (a) or (b);
- wherein said isolated enteropeptidase has a proteolytic activity of cleaving the 4-methylcoumaryl-7-amide (MCA)-substrate Boc-Glu(OBzl)-Ala-Arg-MCA and wherein the amino acid residue positions are numbered according to the amino acid sequence set forth in SEQ ID NO:65.
An isolated enteropeptidase comprising a variant of the amino acid sequence of SEQ ID NO:2, wherein the amino acid at an amino acid residue position selected from the group consisting of position 63, position 105, position 144, position 173 and position 193, is mutated, wherein the isolated enteropeptidase variant is cleavage specific for Asp-Asp-Asp-Asp-Lys (SEQ ID NO:1), and has less proteolytic activity for peptide sequences other than that for SEQ ID NO:1, and wherein the amino acid positions are numbered according to the amino acid sequence set forth in SEQ ID NO:65.
An isolated enteropeptidase comprising the amino acid sequence of SEQ ID NO:4, wherein the isolated enteropeptidase is cleavage specific for Asp-Asp-Asp-Asp-Lys (SEQ ID NO:1), and has less proteolytic activity for peptide sequences other than that for SEQ ID NO:1.
Dependencies are inferred from the claim text.