Production of heterologous peptides

• US 5,834,191 A
• Filed: 02/13/1996
• Issued: 11/10/1998
• Est. Priority Date: 08/13/1993
• Status: Expired due to Term

• Current Assignee: Intrexon Corporation
• Original Assignee: Neugenesis Corporation
• Inventors: Radford, Alan, Parish, John Howard
• Primary Examiner(s): Guzo, David
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/596,300
• Time in Patent Office: 1,001 Days
• Field of Search: 435/6, 435/69.1, 435/91.4, 435/205, 435/254.4, 435/320.1, 536/23.1, 536/24.1, 536/24.33
• US Class Current: 435/6.13
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Abstract

The invention relates to a method and recombinant means for engineering the production of heterologous peptides in filamentous fungus. The invention involves the genetic manipulation of the glucoamylase gene in order to provide a restriction site in same so that the promoter sequence of the glucoamylase gene can be coupled to a sequence encoding a heterologous peptide whereby the production of the peptide will be under the control of the promoter sequence of the glucoamylase gene.

Claims

1. A construct including at least the regulated promoter sequence of the gene encoding the protein glucoamylase of Neurospora crassa and further including at least one restriction site whereby a coding sequence for a heterologous peptide can be introduced into the gene so a heterologous peptide is expressed under the control of said promoter sequence.

2. A construct according to claim 1 wherein the promoter sequence includes an upstream activator comprising the TATA box at position -101 with respect to the start codon ATG shown in FIG. 1 (SEQ ID NO 6).

3. A construct according to claim 1 comprising plasmid pPS8.

4. A construct according to claim 1 comprising the plasmid pGla-Xho I.

5. A construct according to claim 1 comprising the plasmid pGla-Mro I.

6. A construct according to claim 1 comprising the plasmid pGE.

7. A construct according to claim 1 comprising the plasmid pGla XL.

8. A construct according to claim 1 comprising the plasmid pGla XLX.

9. A construct according to claim 1 comprising the plasmid pGLa MXLX.

10. A construct according to claim 1 comprising the plasmid pGS.

11. The regulated promoter of claim 1 which has the DNA sequence structure shown in FIG. 1 (SEQ ID NO:

6).

12. A construct according to claim 1, wherein the promoter sequence includes an upstream activator comprising the CAAT box at position -133 with respect to the start codon ATG shown in FIG. 1 (SEQ ID NO. 6).

13. A construct according to claim 12 wherein the promoter sequence comprises the first 938 nucleotides of the DNA sequence structure shown in FIG. 1 (SEQ ID NO:

6).

14. A construct according to claim 1 wherein the construct further includes a Neurospora-selectable marker.

15. A construct according to claim 14 wherein said marker provides hygromycin-resistance.

16. A construct according to claim 15 wherein said construct further comprises an E. coli-selectable marker.

17. A construct according to claim 16 wherein said marker is a gene encoding ampicillin-resistance.

18. A construct according to claim 1 wherein the construct further comprises DNA sequence structure encoding a secretion signal sequence.

19. A construct according to claim 18 wherein said DNA sequence structure is translationally fused to the codon sequence of a heterologous peptide.

20. A method for transforming filamentous fungus comprising:

(a) introducing the DNA construct of claim 1, linked to a selectable marker, into a filamentous fungal cell; and

(b) culturing said filamentous fungal cell in the presence of an agent to which said selectable marker confers resistance; and

(c) monitoring resistant colonies of said filamentous fungal cells for the production of a heterologous polypeptide;

(d) wherein said filamentous fungal cells are transformed.

21. A method according to claim 20 herein said filamentous fungus is Neurospora crassa.

22. A method for the production of a pre-selected heterologous peptide from at least one filamentous fungus comprising:

a) providing a construct in accordance with claim 20 modified to include a gene encoding a heterologous peptide,

b) transforming a pre-selected species of filamentous fungus with said construct,

c) culturing said transformed fungus; and

d) harvesting the heterologous peptide provided in said construct.

23. A method according to claim 22 wherein said filamentous fungus is Neurospora crassa.

24. A filamentous fungi having inserted therein a construct in accordance with claim 1.

25. A filamentous fungi according to claim 24 wherein said fungi is Neurospora crassa.

26. A primer comprising at least one of the sequence structures shown in FIG. 4, SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12 or SEQ ID NO;

13.