KR910006486A - Fungal expression system - Google Patents

Abstract

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Description

Fungal expression system

As this is a public information case, the full text was not included.

Claims (30)

a) an Aspergillus niger DNA insert of pGW 1800 or pGW 1900, b) a) hybridized with a complete PG II or PG I-coding region made by a DNA insert, galacturonase or polygalactu Structural DNA for a lonase activity polypeptide and optionally a DNA sequence comprising a promoter, signal peptide or leader peptide-coding region and / or transcription terminator, c) a derivative of the DNA sequence as defined in a) or b), or d) a recombinant DNA molecule encoding a complete PG or a signal peptide or a radical peptide thereof and comprising a DNA sequence selected from among those DNA sequences that degenerate within the meaning of the genetic code for the DNA sequence of a), b) or c). The recombinant DNA molecule of claim 1 comprising an Aspergillus Niger DNA insert of pGW 1800 or pGW 1900. The polypeptide and / or signal according to claim 1, which is hybridized with a complete PG II or PG I-coding region made by a DNA insert of pGW 1800 or pGW 1900 and has galacturonase or polygalacturonase activity; A recombinant DNA molecule containing a DNA sequence encoding a leader peptide and / or containing a promoter and / or transcription terminator activity. The method of claim 1, wherein the hybridization is performed with a complete PG II or PG I-coding region made by an Aspergillus Niger DNA insert derivative of pGW 1800 or pGW 1900 or a DNA insert of pGW1800 or pGW 1900, and galacturonase or poly A recombinant DNA molecule comprising a polypeptide having galacturonase activity and / or a derivative of a DNA sequence encoding a signal or leader peptide and / or having a promoter and / or transcription terminator activity. The method of claim 4, wherein λ PG-A9, -A10, -A43, -B4, -B35, -B36, -C1, -C16, -C20, -C37, -D8, -D11, -D12, -E6, A recombinant DNA molecule comprising a vector insert or fragment thereof selected from the group of vectors consisting of -E38, -F5, -G17, -G27, -X31 and -Y33, pGW1756, pGE1910, pGW1800, pGE1900 and pGW1756. The method of claim 4, hybridizing with a complete PG II or PG I-coding region made by one of the DNA fragments or DNA inserts derived from the Aspergillus Niger DNA insert of pGW 1800 or pGW 1900, and the structural gene and / or A recombinant DNA molecule comprising a DNA sequence comprising a sequence encoding a leader or signal peptide and / or having a promoter and / or transcription terminator activity. The method of claim 6, wherein λ PG-A9, -A10, -A43, -B4, -B35, -B36, -C1, -C16, -C20, -C37, -D8, -D11, -D12, -E6, A recombinant DNA molecule comprising a DNA fragment with promoter activity derived from a vector insert selected from the group of vectors consisting of -E38, -F5, -G17, -G27, -X31 and -Y33, pGE1910, pGW1800, pGE1900 and pGW1756. The method of claim 6, wherein λ PG-A9, -A10, -A43, -B4, -B35, -B36, -C1, -C16, -C20, -C37, -D8, -D11, -D12, -E6, A recombinant DNA molecule comprising a DNA peptide and a leader peptide-code derived from a vector insert selected from the group of vectors consisting of -E38, -F5, -G17, -G27, -X31 and -Y33, pGE1910, pGW1800, pGE1900 and pGW1756. The method of claim 6, wherein λ PG-A9, -A10, -A43, -B4, -B35, -B36, -C1, -C16, -C20, -C37, -D8, -D11, -D12, -E6, A recombinant DNA molecule comprising a DNA peptide and a signal peptide-code derived from a vector insert selected from the group of vectors consisting of -E38, -F5, -G17, -G27, -X31 and -Y33, pGE1910, pGW1800, pGE1900 and pGW1756. The method of claim 6, wherein λ PG-A9, -A10, -A43, -B4, -B35, -B36, -C1, -C16, -C20, -C37, -D8, -D11, -D12, -E6, Recombinant DNA molecules comprising DNA fragments with transcription terminator activity derived from a vector insert selected from the group of vectors consisting of -E38, -F5, -G17, -G27, -X31 and -Y33, pGE1910, pGW1800, pGE1900 and pGW1756 . The method of claim 6, wherein λ PG-A9, -A10, -A43, -B4, -B35, -B36, -C1, -C16, -C20, -C37, -D8, -D11, -D12, -E6, Galacturonase or polygalacturonase or galactu, derived from a vector insert selected from the group of vectors consisting of -E38, -F5, -G17, -G27, -X31 and -Y33, pGE1910, pGW1800, pGE1900 and pGW1756 A recombinant DNA molecule comprising a DNA fragment encoding a structural gene for a fragment thereof having lonease or polygalacturonase activity. The recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule is an expression cassette. The recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule is a hybrid vector. The method of claim 13, wherein pGE1800, pGW1900, pGE1756, pGW1910, λPG-A9, λPG-A10, λPG-A43, λPG-B4, λPG-B35, λPG-B36, λPG-C1, λPG-C16, λPG Recombinant selected from the vector group consisting of λPG-C37, λPG-D8, λPG-D11, λPG-D12, λPG-E6, λPG-E38, λPG-F5, λPG-G17, λPG-G27, λPG-X31 and λPG-Y33 DNA molecule. A DNA molecule comprising genes for PG or functional fragments thereof isolated from PG-producing filamentous fungal strains. The DNA molecule of claim 15 isolated from Aspergillus spp. 16. The transcriptional terminator of a PG fragment-coding region or PG gene having a promoter region, signal peptide, leader peptide, prepro-PG, PG or galacturonase or polygalacturonase activity according to claim 15. PGE1800, pGW1900, pGE1756, pGW1910, λPG-A9, λPG-A10, λPG-A43, λPG-B4, λPG-B35, λPG-B36, λPG-C1, λPG-C16, λPG-C20 Hybrid vector selected from the group consisting of -C37, λPG-D8, λPG-D11, λPG-D12, λPG-E6, λPG-E38, λPG-F5, λPG-G17, λPG-G27, λPG-X31 and λPG-Y33 DNA molecule that is a vector insert or fragment thereof. a) isolating genome DNA from suitable fungal cells and screening for expression of the desired polypeptide, for example using a DNA probe or a suitable expression system, or b) isolating mRNA from suitable fungal cells For example, by selecting a target mRNA by hybridizing with a DNA probe or by expressing it in a suitable expression system, screening the expression of the target polypeptide, preparing a single stranded cDNA complementary to the target mRNA, and then preparing a double stranded cDNA therefrom. c) isolating the cDNA from the cDNA library and selecting the desired cDNA, for example using a DNA probe or a suitable expression system, and then screening the expression of the desired polypeptide, and / or d) step a), b) or c Double stranded DNA)) into an appropriate vector, e) transforming the appropriate host cell with the resulting hybrid vector, f) Selecting transformed host cells containing the target DNA from the untransformed host cell or propagating the transformed host cell, if necessary g) separating the target DNA and / or converting the DNA into a mutant or fragment thereof A method for preparing a recombinant DNA molecule of claim 1 or a DNA molecule of claim 15. A host transformed with the recombinant DNA molecule according to claim 1. Suitable host cells are treated with recombinant DNA molecules of the invention, in particular hybrid vectors of the invention, under transfection conditions, optionally in combination with a selection marker gene and optionally selected for transformants. A method of making a converted host. A method for producing a polypeptide, characterized in that the structural gene inserted into the expression cassette according to claim 12 is expressed in a suitable transgenic host according to a conventional method and optionally the polypeptide is separated by a conventional method. The method of claim 21, wherein the Aspergillus niger strain is used as a host. 22. The method of claim 21, wherein the Aspergillus nidulans strain is used as a host. The method of claim 21, wherein the structural gene encodes a hybrid interferon BDBB. The method of claim 21 wherein the structural gene encodes a polypeptide having galacturonase or polygalacturonase activity and is derived from the DNA sequence according to claim 1. A process for preparing a single PG by purifying the single PG from a crude source according to conventional methods. The method of claim 26, wherein the PG is produced in purified form from a crude enzyme mixture produced from Aspergillus Niger. A single galacturonase or polygalacturonase or galacturonase or polygalacturonase activity thereof encoded by a DNA sequence according to claim 1 or a biologically acceptable salt thereof. An enzyme composition comprising the polypeptide according to claim 28 or a mixture thereof, optionally in the form of a premeasured combination with one or more enzymes without PG activity. Use of a polypeptide according to claim 28 or an enzyme composition according to claim 29 in processing plant material. ※ Note: The disclosure is based on the initial application.