Folded recombinant catalytic fragments of multidomain serine proteases, preparation and uses thereof

• US 20050032157A1
• Filed: 08/08/2003
• Published: 02/10/2005
• Est. Priority Date: 08/08/2002
• Status: Active Grant

• Current Assignee: Targetex KFT
• Original Assignee: Targetex KFT
• Inventors: Kardos, Jozsef, Zavodszky, Peter, Gal, Peter, Ambrus-Aikelin, Geza
• Granted Patent: US 7,666,627 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• CPC Class Codes: C07K 1/113 without change of the primary structure
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)

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.

Claims

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.

2. The method of claim 1, wherein the multidomain serine protease encoded by the cDNA insert is a multidomain serine protease of the complement cascade, preferably having the following domain structure CUB-EGF-CUB-CCP1-CCP2-SP and being capable of binding to a recognition molecule of the complement pathway, e.g. to MBL or C1q, wherein said serine protease is of vertebrata, preferably mammalian, more preferably human origin.

3. The method of claim 2, wherein the multidomain serine protease C-terminal fragment encoded by the cDNA insert is a fragment of comprising one or more domain selected from the following domain types:

CCP1, CCP2, SP, preferably, the C-terminal fragment has essentially a domain structure of any of the following;

CCP1-CCP2-SP, CCP2-SP, SP.

4. The method of claim 3, wherein the C-terminal fragment is selected from the following fragments:

MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment, MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3 CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment, C1r CCP1-CCP2-SP fragment (i.e. γ

B-fragment), C1r CCP2-SP fragment or C1r SP fragment, C1s CCP1-CCP2-SP fragment, C1s CCP2-SP fragment or C1s SP fragment.

5. The method of claim 4, wherein the C-terminal fragment comprises on its N-terminal a tag sequence the coding sequence of which increases the efficiency of bacterial protein expression, preferably a sequence suitable for the promoter used for expression.

6. The method of claim 4, wherein said fragment comprises mutation.

7. The method of claim 4, wherein the bacterial host is E. coli and the expression vector is a vector capable of expressing, preferably overexpressing foreign genes in E. coli.

8. The method of claim 1, wherein the step of obtaining the C-terminal fragment in a folded form comprises the following steps:

i) the inclusion bodies formed in the bacterial host are isolated

ii) the protein fragment molecules are renatured from the inclusion bodies,

iii) optionally, the renatured protein fragment molecules are further purified.

9. The method of claim 8, wherein the protein fragment is MASP-fragment, preferably a MASP-fragment selected from the following:

MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment, MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3 CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment, and in the preparation method step c) above comprises the following steps;

i) solubilization of the inclusion bodies,

ii) diluting the solubilized protein fragments and transferring them into a refolding buffer,

iii) allowing the protein fragments to refold at

a pH higher than 7, preferably above pH 8.5, more preferably between 8.5 and 10.5 and

a temperature between 0 and 15°

C.,

in an environment suitable for mildly oxidizing cysteine residues into cystines,

iv) transferring the refolded protein fragments into an appropriate buffer.

10. The method of claim 9, wherein in step i)

solubilization is carried out in an appropriate solubilization agent, e.g. GuHCl or urea, preferably in a buffer comprising 6M GuHCl, any suitable reducing agent e.g. β

-mercaptoethanol or DTT, e.g. 100 mM DTT, and optionally a buffering agent, e.g. 0.1 M Tris-HCl (pH 8.3), e.g. at room temperature or somewhat lower,

the protein concentration in the solution is preferably 0.5 to 25 mg/ml, more preferably 1 to 20 or 2 to 10 mg/ml, even more preferably 3 to 7, e.g. about 5 mg/ml, and in step ii)

the solubilized proteins are preferably diluted directly into the refolding buffer and the dilution is at least 10 fold, preferably at least 100 fold, more preferably 200-600 fold, even more preferably about 400 fold,

prior dilution the refolding buffer is cooled below 10C., preferably below 5°

C., more preferably to about 0°

C. and in step iii),

during refolding, the refolding buffer comprises a redox system, e.g. cysteine/cystine system or a glutathione system wherein the ratio of the reduced and oxidized forms is set to provide an mildly reductive environment,

the refolding buffer further comprises a chaotropic agent in an appropriate concentration, e.g. arginine (0.5 to 1.0 M). In case of MASP-2, GuHCl (0.5 to 2.0 M),

refolding is carried out at a temperature between 0 to 15°

C., preferably between 0 to 10°

C., more preferably between 4 to 8°

C.

11. The method of claim 10, wherein the MASP protein fragment is MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment or MASP-2 CCP2-SP fragment and the pH of the refolding buffer is between pH 8.5 and 9.5, preferably about pH 9.0.

12. The method of claim 10, wherein the MASP protein fragment is MASP-2 SP fragment and the pH is between pH 9.5 to 10.5, preferably pH 10.

13. The method of claim 8, wherein the protein fragment is C1 serine protease fragment, e.g. a C1s or a C1r fragment, and step c) of the preparation method comprises the following steps:

i) solubilization of the inclusion bodies,

ii) diluting the solubilized protein fragments and transferring them into a refolding buffer,

iii) allowing the protein fragments to refold at

a pH higher than 5, preferably between 7 and pH 8.5 and

a temperature between 0 and 1 5°

C.,

in an environment suitable for mildly oxidizing cysteine residues into cystines

iv) transferring the refolded protein fragments into an appropriate buffer.

14. The method of claim 13, wherein in step i)

solubilization is carried out in an appropriate solubilization agent, e.g. GuHCl or urea, preferably in a buffer comprising 6M GuHCl, any suitable reducing agent e.g. β

-mercaptoethanol or DTT, e.g. 100 mM DTT, and optionally a buffering agent, e.g. 0.1 M Tris-HCl (pH 8.3), e.g. at room temperature or somewhat lower,

the protein concentration in the solution is preferably 0.5 to 25 mg/ml, more preferably 1 to 20 or 2 to 10 mg/ml, even more preferably 3 to 7, e.g. about 5 mg/ml, and

In step ii)

the solubilized proteins are diluted directly into the refolding buffer and the dilution is at least 10 fold, preferably at least 100 fold, more preferably 200-600 fold, even more preferably about 400 fold, and in step iii),

during refolding, the refolding buffer comprises a redox system, e.g. cysteine/cystine system or a glutathione system,

the refolding buffer further comprises a chaotropic agent in an appropriate concentration, preferably GuHCl (preferably 2.0 M),

refolding is carried out at a temperature between 0 to 15°

C., preferably between 4 to 15°

C.

23. An isolated C-terminal fragment of a multidomain serine protease of the complement cascade, obtained by the method of any of claims 1 to 14, wherein said fragment is unglycosylated and folded.

24. The isolated fragment of claim 23 wherein the multidomain serine protease is a serine protease capable of binding to a recognition molecule of the complement pathway, e.g. MBL or C1q, said serine protease having preferably the following domain structure CUB-EGF-CUB-CCP1-CCP2-SP, wherein said serine protease is of vertebrata, preferably mammalian, more preferably human origin.

25. The isolated fragment of claim 24 wherein the multidomain serine protease is selected from the following:

MASP-1, MASP-2, MASP-3, C1r, C1s.

26. The isolated fragment of claim 25, wherein the C-terminal fragment is selected from any of the following:

MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment, MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3 CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment, C1r CCP1-CCP2-SP fragment (i.e. γ

B-fragment), C1r CCP2-SP fragment or C1r SP fragment, C1s CCP1-CCP2-SP fragment (i.e. γ

B-fragment), C1s CCP2-SP fragment or C1s SP fragment.

27. The isolated fragment of claim 26, wherein said fragment of the invention comprises on its N-terminal a tag sequence the coding sequence of which increases the efficiency of bacterial protein expression.

28. The isolated fragment of claim 26 which is autoactivated or capable of being autoactivated.

29. The isolated fragment of claim 26 wherein said fragment comprises mutation.

30. The isolated fragment of claim 29 comprising a mutation selected one or more from the following

the activation site of the SP-domain is mutated, e.g. the Arg-Ile bond of the activation site is mutated, preferably to Gln-Ile,

the active Ser is mutated, e.g. to Gly, Ala, Thr, Val, etc., preferably to Ala.

31. Use of any of the C-terminal multidomain complement serine protease fragments defined in any of claims 1 to 30, for raising antibodies.

32. Use of any of the C-terminal multidomain complement serine protease fragments defined in any of claims 1 to 30, as a standard or a control in assessing the level or activity of a multidomain complement serine protease in a biological sample.

33. Use of the C-terminal multidomain complement serine protease fragments defined in any of claims 1 to 30, for screening substrates or inhibitors of MASP-1 or MASP-2 or MASP-3, C1r or C1s.

38. A diagnostic kit for carrying out an assay method of any of claims 34 to 37, said kit comprising a fragment defined in any of claims 1 to 30.

39. Use of a MASP-1 fragment defined in any of claims 1 to 30 for inducing blood coagulation.

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.

16. The expression vector of claim 15 which is a bacterial vector and the said DNA insert is a cDNA insert, preferably encoding a fragment comprising one or more domain selected from the following domain types:

CCP1, CCP2, SP.

17. The expression vector of claim 16, wherein the said cDNA insert encodes a fragment of a multidomain serine protease of the complement cascade (multidomain complement serine protease), preferably a multidomain serine protease selected from the following:

MASP-1, MASP-2, MASP-3, C1r and C1q.

18. The expression vector of claim 17, wherein the c-DNA insert encodes a C-terminal protein fragment having essentially a domain structure of any of the following:

CCP 1-CCP2-SP, CCP2-SP, SP.

19. The expression vector of claim 18, wherein the C-terminal fragment is selected from any of the following:

MASP-1 CCP1-CCP2-SP fragment, MASP-2 CCP1-CCP2-SP fragment, MASP-2 CCP2-SP fragment or MASP-2 SP fragment, MASP-3 CCP1-CCP2-SP fragment, MASP-3 CCP2-SP fragment or MASP-3 SP fragment, C1r CCP1-CCP2-SP fragment (i.e. γ

B-fragment), C1r CCP2-SP fragment or C1r SP fragment, C1s CCP1-CCP2-SP fragment (i.e. γ

B-fragment), C1s CCP2-SP fragment or C1s SP fragment.

20. The expression vector of claim 19 wherein said fragment comprises a mutation.

21. The expression vector of claim 16 wherein the cDNA insert comprises a sequence capable of increasing the efficiency of bacterial protein expression, said sequence being operably linked to the sequence encoding the protein fragment.

22. A vector of any of the claims 15 to 21, wherein the means for expressing the DNA insert sequence encoding any of the protein fragments as disclosed above, operably linked to a promoter, preferably an inducible overexpressing promoter capable of driving expression in a suitable bacterial host, preferably, the bacterial host is E. coli and the promoter is a promoter driving overexpression.

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.

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.

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.

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.

40. Use of an inhibitor of MASP-1, preferably α

2M or C1-inhibitor, for reducing blood coagulation.

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.