Folded recombinant catalytic fragments of multidomain serine proteases, preparation and uses thereof
First Claim
1. Recombinant method for the preparation of an unglycosylated folded C-terminal fragment of a multidomain serine protease, comprising the following steps:
- a bacterial expression vector for expressing a DNA insert encoding a C-terminal fragment of a multidomain serine protease is provided, said C-terminal fragment is produced in a bacterial host by using the said vector and obtained in a folded form from the bacterial host.
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Abstract
The invention relates to unglycosylated folded C-terminal fragments of a multidomain serine protease of the complement cascade obtainable by expression in a bacterial host, wherein said serine protease is capable of binding a recognition molecule of the complement cascade, e.g. C1 or MBL. The invention also relates to methods and bacterial expression vectors for the preparation of said fragments, uses of said fragments for raising antibodies and screening substrates or inhibitors of said serine proteases and uses of the fragments in research and treatment of complement related disorders. The invention also relates to assay methods for assessing MASP-1 and MASP-2 levels in a sample of biological origin. The invention provides for research tools, assays and diagnostic kits useful in complement research and research and diagnosis of complement related disorders.
43 Citations
41 Claims
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1. Recombinant method for the preparation of an unglycosylated folded C-terminal fragment of a multidomain serine protease, comprising the following steps:
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a bacterial expression vector for expressing a DNA insert encoding a C-terminal fragment of a multidomain serine protease is provided, said C-terminal fragment is produced in a bacterial host by using the said vector and obtained in a folded form from the bacterial host. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 38, 39)
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- 15. A prokaryotic expression vector, said vector comprising a DNA insert encoding a C-terminal fragment of a multidomain serine protease and means for expressing said fragment in a bacterial host, wherein said serine protease is of vertebrata, preferably mammalian, more preferably human origin.
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34. An assay method for measuring the level of a multidomain complement serine protease in a biological sample, wherein the presence of said serine protease is quantitatively detected in the sample by a labeled monoclonal antibody and the obtained signal is compared with a signal obtained for a control sample comprising a respective complement protease fragment according the invention.
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35. An assay method for measuring the activity of a multidomain complement serine protease in a biological sample, wherein the activity on a substrate of said protease is measured, and an appropriate fragment, preferably a CCP1-CCP2-SP fragment, of said protease according to the invention is used as a standard with the proviso that it has the same specific activity as the respective protease or the ratio of the activities of the native protease and the fragment is known.
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36. An assay method for assessing MASP-1 and MASP-2 levels in a sample of biological origin, said method comprising
monitoring C2 cleavage and C4 cleavage by MASP proteins in aliquots of the sample whereas, if desired, other complement pathways are blocked, considering C4 conversion as a result of MASP 2 activity and C2 conversion as a result of MASP-1 and MASP-2 activity together calculating MASP-1 and MASP-2 levels using either known specific activity values of said proteins or MASP-1 and MASP-2 CCP1-CCP2-SP fragments, respectively, as inner standards.
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37. An assay method for assessing MASP-1 and MASP-2 levels in a sample of biological origin, said method comprising
monitoring C2 cleavage in the sample and considering C2 conversion as a result of MASP-1 and MASP-2 activity together, adding a calculated amount of α - 2M to the sample to inhibit MASP-1 activity but leaving MASP-2 activity unchanged or changing it to a negligible or a calculable extent,
monitoring C2 activity in the sample comprising α
2M,calculating MASP-1 and MASP-2 levels using either known specific activity values of said proteins or MASP-1 and MASP-2 CCP1-CCP2-SP fragments, respectively, as inner standards.
- 2M to the sample to inhibit MASP-1 activity but leaving MASP-2 activity unchanged or changing it to a negligible or a calculable extent,
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40. Use of an inhibitor of MASP-1, preferably α
- 2M or C1-inhibitor, for reducing blood coagulation.
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41. Method for treatment of a patient in need of inhibiting complement activity exerted through the lectin pathway, preferably reperfusion injury, comprising administering any inhibitor of the lectin complement pathway, preferably an inhibitor of MASP-2, to the patient in an effective quantity.
Specification