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Novel fungal proteins and nucleic acids encoding same

A protein, nucleic acid molecule technology, applied in the field of recombinant production of the nucleic acid and peptidase

Active Publication Date: 2006-11-29
AMYRA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The activity of aminopeptidase, which can also play a role in fungal infection, has long been confirmed by (De Bersaques & Dockx, Arch. Belg. Dermatol. Syphiligr. 29: 135-140 (1973); Danew & Friedrich, Mykosen 23: 502- 511(1980)) mycelium and culture medium, however, no aminopeptidase or carboxypeptidase has been isolated and characterized from dermatophytes

Method used

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  • Novel fungal proteins and nucleic acids encoding same
  • Novel fungal proteins and nucleic acids encoding same
  • Novel fungal proteins and nucleic acids encoding same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0260] Example 1: Materials and methods

[0261] Strains and plasmids

[0262] A clinical isolate of Trichophyton rubrum, CHUV 862-00, was used in this study. Bacteriophage lambda EMBL3 (Promega, Wallisellen, Switzerland) was propagated with E. coli LE392. All plasmid-subcloning experiments were performed in E. coli DH5α using the plasmid pMTL2I (Chambers et al., Gene 68:139-149 (1988)). The recombinant peptidase was expressed using P. pastoris GSI15 and the expression vector pKJ 113 (Borg-Von Zepelin et al, Mol. Microbiol 28:543-554 (1998)). It is well known in the art that P. pastoris can be used to express a large number of recombinant proteins.

[0263] Trichophyton rubrum growth medium

[0264] Trichophyton rubrum was grown on Sabouraud agarose or liquid medium (Bio-Rad, Munchen, Germany), or in order to increase the proteolytic activity of the product, in a medium containing 0.2% soybean protein (Supro 1711, Protein Technology International, St. Lauise. MO) as the s...

Embodiment 2

[0308] Example 2: Hydrolase Activity of Trichophyton rubrum Secreted Proteins

[0309] Trichophyton rubrum was cultured at 30°C on a medium containing 0.2% soybean protein as the sole nitrogen and carbon source. After 14 days of growth, accompanied by clarification of the medium was recorded, real proteolytic enzyme activity (400 U / ml) was detected using resorufin-labeled casein as substrate. Its proteolytic activity was inhibited by 15% and 85% by PMSF and o-phenanthroline, respectively, demonstrating that Trichophyton rubrum secretes serine and metalloproteases. Western blot analysis of the culture supernatant revealed that Trichophyton rubrum was similar to Microsporum canis, secreting endogenous proteases of the subtilisin family (MEROPS>S8) and fungalysin family (MEROPS>M36), similar to A .oryzae secretes alkaline phosphatase ALP and neutral metalloprotease NPI (see figure 1 ). In addition, high activities of substrates such as Leu-AMC and Leu-pNA were detected in the ...

Embodiment 3

[0310] Example 3: Aminopeptidase activity of Trichophyton rubrum secretions

[0311] The nucleotide sequence of the M. canis endoprotease gene is 50-70% similar to the homologous gene. These genes encode subtilisin and fungalysin secreted by A. oryzae and A. fumigtus. In addition, the genes of M. canis and Aspergillis have a linear correspondence of intron-exon structure. Therefore, the DNA sequences of genes encoding aminopeptidases from A. oryzae and yeast were used to design probes for screening T. rubrum genomic DNA libraries. Using the recombinant protein, by comparison with the protein secreted by the opportunistic pathogen Aspergillus fumigatus. Characterization of aminopeptidase secreted by Trichophyton rubrum.

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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.

Description

technical field [0001] The present invention relates to a novel polypeptide enzyme with unique catalytic properties and its encoding nucleic acid. Specifically, the present invention relates to a nucleic acid encoding a new leucine aminopeptidase and other amino or carboxypeptidases, specifically referred to as EXOX in the present invention, and also includes vectors, host cells, antibodies, and the production of the nucleic acid and recombinant methods of peptidases. The gene can be obtained from the following two fungi: Trichophyton rubrum, Aspergillus fumigatus. Background technique [0002] Bacteria, yeast and filamentous fungi, as well as some special plant cells, invertebrates and vertebrates all express a membrane protein that is useful for absorbing amino acids, dipeptides or tripeptides (Lubkowitz, et al., Microbiology 143:387-396( 1997); Hauser et al., Mol. Membr. Biol. 18(1): 105-112 (2001); Stacey et al., Trends Plant Sci. 7(6): 257-263 (2002); Rubio-Aliaga & D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N9/48C12N5/10A61K38/46A61K31/00A61K33/00G01N33/50A61K38/00C12P21/06C12Q1/18C12Q1/37
CPCC07K14/37A61K38/00C12Q1/37G01N2333/37C12Q1/18C12N9/48C12P21/06A61P1/00A61P1/14A61P31/10A61P37/08A61P43/00
Inventor 米歇尔·莫诺雷托·斯托克林埃里克·格罗茨曼
Owner AMYRA BIOTECH
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