Chimeric serine proteases
First Claim
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1. A method for determining whether a substance has serine protease inhibiting activity, comprising:
- a) combining a chimeric protein with a target substance under conditions such that the chimeric protein exhibits serine protease activity in the absence of the target substance, wherein the chimeric protein comprises a first sequence and a second sequence C-terminal to the first sequence and linked to the first sequence by one or more peptide bonds, the first sequence having the amino acid sequence of the first catalytic domain half of a first serine protease and the second sequence having the amino acid sequence of the second catalytic domain half of a second serine protease different from the first serine protease;
wherein the first sequence has the amino acid sequence of the first catalytic domain half of factor Xa and the second sequence has the amino acid sequence of the second catalytic domain half of trypsin;
b) measuring the serine protease activity of the chimeric protein in the presence of the target substance and in the absence of the target substance, to detect whether the serine protease activity of the chimeric protein in the presence of the target substance is decreased relative to the serine protease activity of the chimeric protein in the absence of the target substance; and
c) if a decrease in serine protease activity is detected, determining that the target substance has serine protease inhibiting activity.
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Abstract
A chimeric serine protease whose protease domain is composed of two domain halves (half-sides) with a β-folded sheet structure, wherein the first domain half corresponds to the first domain half of a first serine protease and the second domain half corresponds to the second domain half of a second serine protease, has improved properties and can be readily crystallized.
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Citations
5 Claims
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1. A method for determining whether a substance has serine protease inhibiting activity, comprising:
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a) combining a chimeric protein with a target substance under conditions such that the chimeric protein exhibits serine protease activity in the absence of the target substance, wherein the chimeric protein comprises a first sequence and a second sequence C-terminal to the first sequence and linked to the first sequence by one or more peptide bonds, the first sequence having the amino acid sequence of the first catalytic domain half of a first serine protease and the second sequence having the amino acid sequence of the second catalytic domain half of a second serine protease different from the first serine protease;
wherein the first sequence has the amino acid sequence of the first catalytic domain half of factor Xa and the second sequence has the amino acid sequence of the second catalytic domain half of trypsin;
b) measuring the serine protease activity of the chimeric protein in the presence of the target substance and in the absence of the target substance, to detect whether the serine protease activity of the chimeric protein in the presence of the target substance is decreased relative to the serine protease activity of the chimeric protein in the absence of the target substance; and
c) if a decrease in serine protease activity is detected, determining that the target substance has serine protease inhibiting activity. - View Dependent Claims (2, 3, 4, 5)
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Specification
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Current AssigneeBoehringer Mannheim GmbH (Roche Holding AG)
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Original AssigneeBoehringer Mannheim GmbH (Roche Holding AG)
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InventorsHopfner, Karl-Peter, Huber, Robert, Kopetzki, Erhard, Engh, Richard, Bode, Wolfram
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Primary Examiner(s)HUTSON, RICHARD G
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Application NumberUS09/664,595Time in Patent Office1,177 DaysField of Search435/219.23, 435/440, 435/69.1, 536/23.1, 536/23.2, 536/23.4, 530/350, 530/380US Class Current435/23CPC Class CodesC07K 2319/00 Fusion polypeptideC12N 9/6424 Serine endopeptidases (3.4.21)C12N 9/6427 Chymotrypsins (3.4.21.1; 3....C12N 9/6432 Coagulation factor Xa (3.4....C12N 9/6467 Granzymes, e.g. granzyme A ...C12Y 304/21006 Coagulation factor Xa (3.4....
