Novel fungal proteins and nucleic acids encoding same

• US 20070009988A1
• Filed: 08/25/2004
• Published: 01/11/2007
• Est. Priority Date: 08/25/2003
• Status: Active Grant

• Current Assignee: AMYRA Biotech AG
• Original Assignee: Funzyme Biotechnologies SA
• Inventors: Grouzmann, Eric, Monod, Michel, Stocklin, Reto
• Granted Patent: US 7,468,267 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/14 Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/37 from fungi
• CPC Class Codes: C12N 9/48 acting on peptide bonds (3.4)
• 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
• CPC Class Codes: C12Q 1/18 Testing for antimicrobial activity of a material
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/37 from fungi

Abstract

Disclosed herein are fungal nucleic acid sequences that encode novel polypeptides. Also disclosed are polypeptides encoded by these nucleic acid sequences, as well as derivatives, variants, mutants, or fragments of the aforementioned polypeptide, polynucleotide, or antibody. The novel leucine aminopeptidase (LAP) and other amino- and carboxypeptidases polypeptides, referred to herein as EXOX nucleic acids and proteins of the invention are useful in a variety of medical, research, and commercial applications.

Claims

1. (canceled)

2. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

2, 3, 6, 21, 24, 27, 30, and 35.

15. An isolated nucleic acid molecule encoding the polypeptide of claim 2.

3-14. -14. (canceled)

16-41. -41. (canceled)

42. An enzyme cocktail comprising

a) a dipeptidyl peptidase IV (DPP IV) comprising SEQ ID NO:

35, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 90% of identity, and

b) an aminopeptidase originating from a dermatophyte comprising the mature form of an amino acid sequence comprising either

i) a leucine aminopeptidase (LAP) comprising SEQ ID NO;

3, a biologically active fragment thereof, a naturally occurring allelic variant thereof, and/or a sequence having at least 90% of identity;

or

ii) a leucine aminopeptidase (LAP) comprising SEQ ID NO;

6, a biologically active fragment thereof, a naturally occurring allelic variant thereof, and/or a sequence having at least 90% of identity.

43. The enzyme cocktail of claim 42, comprising the aminopeptidase of (i) and the aminopeptidase of (ii).

44. The enzyme cocktail of claim 42, characterized in that the dermatophyte is selected from the group consisting of Epidermophyton floccosum, Microsporum audouinii, Microsporum ferrugineum, Trichophyton concentricum, Trichophyton kanei, Trichophyton megninii, Trichophyton mentagrophytes, Trichophyton raubitschekii, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton soudanense, Trichophyton tonsurans, Trichophyton violaceum, Trichophyton yaoundei, Microsporum canis, Microsporum equinum, Microsporum nanum, Microsporum persicolor, Trichophyton equinum, Trichophyton simii, Trichophyton verrucosum, Microsporum gypseum, Trichophyton ajelloi, and Trichophyton terrestre.

45. The enzyme cocktail of claim 42, further comprising one or more proteases.

46. The enzyme cocktail of claim 45, wherein said one or more proteases are selected from the group consisting of trypsin, pronase, chymotrypsin, and proteinaseK.

47. The enzyme cocktail of claim 42, characterized in that the leucine aminopeptidase (LAP) is ruLAP2.

48. The enzyme cocktail of claim 42, for use in the degradation, hydrolysis or maturation of polypeptide substrates or proteins.

49. A method of using the enzyme cocktail of claim 42, in an activity selected from the group consisting of degradation of by-products, toxic or contaminant proteins;

degradation of prions or viruses;

degradation of proteins for proteomics;

degradation of cornified substrate;

hydrolysis of polypeptides for amino acid analysis;

wound cleaning;

wound healing;

cosmetology prothesis cleaning and/or preparation;

fabric softeners;

soaps;

cleaning or disinfection of septic tanks;

cleaning of disinfection of a container containing proteins that should be removed or sterilized; and

cleaning of surgical instruments.

50. The method of claim 49, wherein the cosmetology method is selected from the group consisting of peeling tools, depilation, dermabrasion and dermaplaning.

51. A kit comprising, in one or more containers, the enzyme cocktail of claim 42.

56. A method of degrading a polypeptide substrate, the method comprising bringing the polypeptide substrate into contact with the enzyme cocktail of claim 42.

57. The method of claim 56, wherein said enzyme cocktail sequentially digests a full-length polypeptide substrate or a full-length protein.

58. The method of claim 56, characterized in that the polypeptide substrate is selected from the group consisting of casein, gliadin, gluten, bovine serum albumin or fragments thereof.

59. The method of claim 56, wherein the polypeptide substrate length is from 2 to 200 amino acids.

60. A method of removing amino acids from the amino terminus of a protein, comprising bringing said protein into contact with the enzyme cocktail of claim 42.

61. The method of claim 60, characterized in that the amino terminus of said protein comprises a His tag.

52. A method of treating a pathological state in a mammal, comprising administering to the mammal an enzyme cocktail in an amount that is sufficient to alleviate the pathological state, wherein the enzyme cocktail comprises

a) a dipeptidyl peptidase IV (DPP IV) comprising SEQ ID NO:

35, a biologically active fragment thereof, a naturally occurring allelic variant thereof, or a sequence having at least 90% of identity, and

b) an aminopeptidase originating from a dermatophyte comprising the mature form of an amino acid sequence comprising either

i) a leucine aminopeptidase (LAP) comprising SEQ ID NO;

3, a biologically active fragment thereof, a naturally occurring allelic variant thereof, and/or a sequence having at least 90% of identity;

or

ii) a leucine aminopeptidase (LAP) comprising SEQ ID NO;

6, a biologically active fragment thereof, a naturally occurring allelic variant thereof, and/or a sequence having at least 90% of identity.

53. The method of claim 52, wherein the enzyme cocktail comprises both the aminopeptidase of (i) and the aminopeptidase of (ii).

54. The method of claim 52, wherein the pathological state is selected from the group consisting of celiac disease, digestive tract malabsorption, an allergic reaction, an enzyme deficiency, a fungal infection, mycoses and sprue.

55. The method of claim 54, wherein the allergic reaction is a reaction to gluten or fragments thereof.

62. A composition for removing amino acids from the amino terminus of a protein comprising a dipeptidyl peptidase IV (DPPIV) of SEQ ID NO:

35, a biologically active fragment thereof, a naturally occurring allelic variants thereof, or a sequence having at least 90% of identity.

63. The composition of claim 62, characterized in that the amino terminus of said protein comprises a Xaa-Pro tag.

64. The composition of claim 63, characterized in that Xaa is an amino acid comprising at least two vicinal nucleophilic groups.

65. The composition of claim 64, characterized in that said amino acid is serine, threonine or cysteine.

66. A method for recovering a protein in its mature form without a di-peptide tag, comprising the steps of:

(1) purification by covalent capture and,

(2) removal of the di-peptide tag with the composition of claim 62.