METHOD FOR THE PREPARATION OF SURFACTANT PEPTIDES

• US 20130303726A1
• Filed: 04/15/2013
• Published: 11/14/2013
• Est. Priority Date: 04/17/2012
• Status: Abandoned Application

• Current Assignee: Chiesi Farmaceutici SPA (Valline Srl)
• Original Assignee: Chiesi Farmaceutici SPA (Valline Srl)
• Inventors: MOZZARELLI, Andrea, Raboni, Samanta, Pioselli, Barbara
• Application Number:
  US13/862,590
• Publication Number:
  US 20130303726A1
• Time in Patent Office: Days
• US Class Current: 530/300
• CPC Class Codes: C07K 14/785 Alveolar surfactant peptides; Pulmonary surfactant peptides
• CPC Class Codes: C07K 2319/24 containing a MBP (maltose binding protein)-tag
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

Abstract

Surfactant-protein C peptides (SP-C peptides) may be prepared by the heterologous expression of a fusion protein of an SP-C peptide and a maltose binding protein.

Claims

1. An expression cassette, comprising:

(a) a polynucleotide sequence encoding an SP-C peptide;

(b) a polynucleotide sequence encoding an MBP protein; and

(c) a polynucleotide sequence encoding a linker peptide,

wherein said a polynucleotide sequence encoding a linker peptide encodes a protease cleavage site and is located between said polynucleotide sequence encoding an SP-C peptide and said polynucleotide sequence encoding an MBP protein, and

said encoding polynucleotide sequence being operatively linked to a promoter sequence suitable for the expression in a prokaryotic cell.

2. An expression cassette according to claim 1, wherein said SP-C peptide is SP-C33(Leu) and has the sequence shown in SEQ ID NO:

1.

3. An expression cassette according to claim 1, wherein the MBP protein has the sequence shown in SEQ ID NO:

2.

4. An expression cassette according to claim 1, wherein said polynucleotide sequence encoding an SPC peptide has the sequence shown in SEQ ID NO:

3.

5. An expression cassette according to claim 1, wherein said polynucleotide sequence encoding an MBP protein has the sequence shown in SEQ ID NO:

4.

6. An expression cassette according to claim 1, wherein said linker peptide has the amino acid sequence shown in SEQ ID NO:

5.

7. An expression cassette according to claim 1, wherein said polynucleotide sequence encoding a linker peptide has the sequence shown in SEQ ID NO:

5.

8. An expression cassette according to claim 1, wherein said polynucleotide sequence encoding an MBP protein is located at the N-terminus.

9. An expression cassette according to claim 1, wherein said polynucleotide sequence encoding an SP-C peptide is located at the C-terminus.

10. An expression cassette according to claim 1, wherein said polynucleotide sequence encoding an MBP protein is located at the N-terminus and said polynucleotide sequence encoding an SP-C peptide is located at the C-terminus.

11. An expression cassette according to claim 1, wherein said promoter is an inducible tac promoter.

12. An expression vector, containing an expression cassette according to claim 1.

13. An expression vector according to claim 12, which is a plasmid.

14. An expression vector according to claim 13, which is a pBR32-based plasmid.

15. A method for preparing an SP-C peptide, which comprises:

(i) providing a vector carrying an expression cassette according to claim 1;

(ii) introducing said vector into a prokaryotic cell and maintaining said cell under conditions permissive for the expression of the fusion protein encoded by said casette;

(iii) purifying said fusion protein;

(iv) cleaving said fusion protein with a suitable protease; and

(v) isolating the SP-C peptide.

16. A method according to claim 15, wherein said prokaryotic cell is an E. coli bacterial cell.

17. A method according to claim 15, wherein said purifying of said fusion protein is carried out by affinity chromatography using a MBP ligand-bound resin.

18. A method according to claim 15, wherein an enterokinase protease is used for said cleaving said fusion protein.

19. An SP-C peptide, which is prepared by a method according to claim 15.

20. A pulmonary surfactant composition, which comprises an SP-C according to claim 19.