Sushi peptide multimer

• US 7,763,704 B2
• Filed: 07/02/2004
• Issued: 07/27/2010
• Est. Priority Date: 07/04/2003
• Status: Active Grant

• Current Assignee: National University of Singapore
• Original Assignee: National University of Singapore
• Inventors: Ding, Jeak Ling, Ho, Bow
• Primary Examiner(s): DUFFY, PATRICIA ANN
• Application Number:
  US10/563,551
• Publication Number:
  US 20080113906A1
• Time in Patent Office: 2,216 Days
• Field of Search: None
• US Class Current: 530/300
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C12N 9/6408 Serine endopeptidases (3.4.21)
• CPC Class Codes: C12Y 304/21084 limulus clotting factor C (3.4.21.84)
• CPC Class Codes: Y10S 435/975 Kit
• CPC Class Codes: Y10S 530/81 Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier
• CPC Class Codes: Y10S 930/28 Bound to a nonpeptide drug, nonpeptide label, nonpeptide carrier, or a nonpeptide resin

Abstract

Endotoxin, also known as lipopolysaccharides (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, binds and neutralizes LPS activity. Fluorescent tagged-S3 is shown to detect LPS-containing bacteria. For large-scale production of S3 and to mimic other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in E. coli. Tetramer of S3 for example is shown to display an enhanced inhibitory effect on LPS-induced activities. An affinity matrix based on tetramer of S3 is also shown to be particularly efficient at removing LPS.

Claims

1. A polypeptide comprising more than one S3 peptides comprising the amino acid sequence set forth in SEQ ID No:

1, wherein at least two of the S3 peptides are separated by a linking sequence cleavable by a protease.

2. The polypeptide of claim 1 wherein the S3 peptides are in tandem repeat.

3. The polypeptide of claim 1 comprising 2 to 10 S3 peptides.

4. The polypeptide of claim 1 comprising two S3 peptides.

5. The polypeptide of claim 1 comprising three S3 peptides.

6. The polypeptide of claim 1 comprising four S3 peptides.

7. The polypeptide of claim 1 comprising eight S3 peptides.

11. The polypeptide of claim 1 wherein the more than one S3 peptide is tagged with a detectable label.

12. The polypeptide of claim 11 wherein the label is detectable by fluorescence.

13. The polypeptide of claim 1 immobilized on a solid medium.

14. The polypeptide of claim 11 immobilized on a solid medium.

15. The polypeptide of claim 14 wherein the solid medium is agarose.

16. A package comprising the polypeptide of claim 1 and instructions for its use in detecting LPS-containing bacteria in a sample.

17. A package comprising the polypeptide of claim 13 and instructions for its use for removing LPS or LPS-containing bacteria from a sample.

8. A polypeptide comprising more than one S3 peptides comprising the amino acid sequence set forth in SEQ ID No:

1, wherein at least two of the S3 peptides are separated by a linking sequence cleavable by acid digestion.

9. The polypeptide of claim 8 wherein at least one of the linking sequences comprises Asp-Pro.

10. A polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

9, having a linking sequence cleavable by a protease.