Trichoderma reesei host cells expressing a glucoamylase from aspergillus fumigatus and methods of use thereof
A host cell, Trichoderma reesei technology, applied in the direction of biochemical equipment and methods, glycosylase, the use of vectors to introduce foreign genetic material, etc.
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[0220] 3. Preparation of AfGA and its variants
[0221] AfGA or a variant thereof can be isolated from the host cell, eg, by secretion of the AfGA or variant from the host cell. Cultured cell material comprising AfGA or a variant thereof may be obtained following secretion of AfGA or a variant thereof from a host cell. AfGA or variants thereof are optionally purified prior to use. The AfGA gene can be cloned and expressed according to methods well known in the art. Suitable host cells include bacterial, plant or yeast cells, such as filamentous fungal cells. Particularly useful host cells include Trichoderma reesei. T. reesei host cells express AfGATR at higher or at least comparable levels compared to naturally expressed AfGA A. fumigatus.
[0222] In some embodiments, AfGA is heterologously expressed in the host at at least 10 g / L. In some embodiments, AfGA is heterologously expressed at at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, or 110 ...
example 1
[0432] Example 1: Cloning of AfGA1 .
[0433] Genomic DNA of A. fumigatus Af293 was purchased from the Fungal Genetics Stock Center, Kansas City, MO (FGSC A1100). The genome of Aspergillus fumigatus was sequenced. The nucleotide sequence of the AfGA1 gene (within the disclosed genome in NCBI reference sequence NC_007195), and the predicted glucan 1,4-alpha-glucosidase (NCBI accession number XP_749206) encoded by the AfGA1 gene were obtained in the NCBI database ) amino acid sequence. AfGA1 is homologous to other fungal glucoamylases as determined from BLAST searches. See Figure 1. The nucleotide sequence of the AfGA1 gene contains three introns, and the nucleotide sequence is shown in SEQ ID NO:8.
[0434] The AfGA1 gene was amplified from genomic DNA of Aspergillus fumigatus using the following primers: Primer 1: AfGA1-Fw 5'-GCG GCGGCCGC ACC atgcctcgcctttcctacgc-3' (SEQ ID NO: 9), and Primer 2: AfGA1-Rv 5'-cc ggcgcgccc TTA tcactgccaagtatcattctcg-3' (SEQ ID NO: 10). Th...
example 2
[0435] Example 2: Expression and purification of AfGA1TR .
[0436] The plasmid pJG222(Trex3gM-AfGA1) was transformed into a quadruple deletion Trichoderma reesei strain (described in WO 05 / 001036) using the gene gun method (Te'o et al., J. Microbiol. Methods ("Journal of Microbiology Methods") "), 51:393-99, 2002). Transformed colonies (about 50) appeared within about 1 week. After 5 days of growth on acetamide plates, colonies were inoculated in 250 ml shake flasks containing 30 ml glucose / agarose defined medium for protein expression. The protein AfGA1TR was secreted into the extracellular medium, and the filtered medium was used for SDS-PAGE and glucoamylase activity assay on DP7 to confirm enzyme expression.
[0437] The stable strains were then grown in defined medium in 7L fermentors. The fermentation broth was collected by centrifugation. After centrifugation, filtration and concentration, a 450 ml concentrated sample was obtained. By using the BCA method (Prote...
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