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

• US 7,666,627 B2
• Filed: 08/08/2003
• Issued: 02/23/2010
• Est. Priority Date: 08/08/2002
• Status: Active Grant

• Current Assignee: Targetex KFT
• Original Assignee: Targetex KFT
• Inventors: Gál, Péter, Závodszky, Péter, Ambrus-Aikelin, Géza, Kardos, József
• Primary Examiner(s): Desai; Anand U
• Assistant Examiner(s): Moore; William W
• Application Number:
  US10/636,602
• Publication Number:
  US 20050032157A1
• Time in Patent Office: 2,391 Days
• Field of Search: None
• US Class Current: 435/69.7
• 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. A recombinant method for the preparation of an unglycosylated folded C-terminal fragment of a multidomain serine protease of the vertebrate complement cascade, wherein said multidomain serine protease is MASP-1, said method comprising the following steps:

i) inserting into a bacterial expression vector a DNA sequence encoding a C-terminal fragment of a vertebrate MASP-1 serine protease and wherein the encoded C-terminal MASP-1 protein fragment consists of the domain structure CCP1-CCP2-SP fused at its amino terminus to a peptide amino acid sequence of either SEQ ID NO;

19 or SEQ ID NO;

20,

ii) transforming a bacterial host cell with said expression vector,

iii) inducing the expression of the MASP-1 C-terminal fragment consisting of the domain structure CCP1-CCP2-SP fused at its amino terminus to a peptide of SEQ ID NO;

19 or SEQ ID NO;

20,

iv) isolating inclusion bodies comprising the unglycosylated MASP-1 C-terminal fragment fusion protein of step (iii) consisting of the domain structure CCP1-CCP2-SP fused at its amino terminus to a peptide of SEQ ID NO;

19 or SEQ ID NO;

20 from the bacterial host cell,

v) renaturing the MASP-1 C-terminal fragment fusion protein of step (iv) to provide unglycosylated re-folded MASP-1 C-terminal fragment fusion protein, and

vi) purifying the unglycosylated folded MASP-1 C-terminal fragment fusion protein.

2. A bacterial expression vector comprising an inserted DNA sequence encoding a C-terminal fragment of a vertebrate multidomain serine protease of the complement cascade wherein said multidomain serine protease is MASP-1 and the encoded C-terminal MASP-1 protein fragment consists of the domain structure CCP1-CCP2-SP fused at its amino terminus to a peptide amino acid sequence of SEQ ID NO:

19 or SEQ ID NO;

20.

3. An isolated, unglycosylated, and folded, C-terminal fragment of a vertebrate MASP-1 serine protease that has the domain structure CCP1-CCP2-SP and is fused at its amino terminus to a peptide consisting only of SEQ ID NO:

19 or SEQ ID NO;

20.

4. The isolated fragment of claim 3 which is autoactivated or capable of being autoactivated.

5. The isolated fragment of claim 3 wherein said fragment comprises a mutation selected from either or both of the following

the activation site of the SP-domain is mutated to prevent autoactivation,

the active Ser is mutated.