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WO1997013853A2 - Detection de proteines - Google Patents

Detection de proteines Download PDF

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Publication number
WO1997013853A2
WO1997013853A2 PCT/EP1996/004510 EP9604510W WO9713853A2 WO 1997013853 A2 WO1997013853 A2 WO 1997013853A2 EP 9604510 W EP9604510 W EP 9604510W WO 9713853 A2 WO9713853 A2 WO 9713853A2
Authority
WO
WIPO (PCT)
Prior art keywords
ser
gly
ala
thr
leu
Prior art date
Application number
PCT/EP1996/004510
Other languages
English (en)
Other versions
WO1997013853A3 (fr
Inventor
Henriëtte Catharina VAN DEN BROECK
Leendert Hendrik De Graaff
Jacob Visser
Albert Johannes Joseph Van Ooyen
Original Assignee
Gist-Brocades B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Gist-Brocades B.V. filed Critical Gist-Brocades B.V.
Priority to AU72943/96A priority Critical patent/AU7294396A/en
Priority to EP96934722A priority patent/EP0796328A2/fr
Priority to JP9514730A priority patent/JPH10510720A/ja
Publication of WO1997013853A2 publication Critical patent/WO1997013853A2/fr
Publication of WO1997013853A3 publication Critical patent/WO1997013853A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)

Definitions

  • the DNA of interest is identified by the expression of the protein it encodes, rather than by going through the tedious tasks of purifying the protein, amino acid sequencing and molecular cloning of o the gene.
  • One technique of expression cloning is a plate assay, wherein a cDNA library is plated onto a medium of a composition which enables screening for positive colonies. Screening for the protein of interest does not require pure protein, only the availability of a suitable assay for the detection of activity. This may be a standard assay if for example, cellulase activity may be detected using an overlay containing carboxymethyl cellulose, followed by visualisation by staining with Congo Red.
  • the nucleic acid sequence of the invention is operably linked to a promoter capable of expressing the sequence.
  • "Operably linked” refers to a juxtaposition wherein the promoter and the nucleic acid sequence encoding the polypeptide or protein are in a relationship permitting the coding sequence to be expressed under the control of the promoter.
  • there may be elements such as 5' non-coding sequence between the promoter and coding sequence.
  • Such sequences can be included in the vector if they enhance or do not impair the correct control of the coding sequence by the promoter.
  • the invention also provides polypeptides encoded by the nucleic acids of the invention.
  • A. niger N400 cultures were grown for 69 and 81 h respectively, as described in s EP-A- 0 463 706 but without yeast extract and with 2% of a crude wheat arabinoxylan fraction instead of oat spelt xylan, after which the mycelium was harvested by filtration and then washed with sterile saline. The mycelium was subsequently frozen in liquid nitrogen after which it was powdered using a Microdismembrator (Braun). Total RNA was isolated from mycelial powder in accordance with the guanidium thiocyanate/CsCI o protocol described in Sambrook et al. (1 989), except that the RNA was centrifuged twice using a CsCl gradient.
  • the recombinant Uni-ZAP XR clones containing A. niger cDNA were converted to Bluescript phagemids using superinfection with the filamentous helper phage EXASSIST'TM and E.coli SOLR strain which are included in the cDNA synthesis kit from Stratagene, according to the manufacturer's instructions.
  • a glycerol stock containing about 100 colonies per vl of suspension was stored at -80°C.
  • Cells are plated in an overlay of 5 ml containing about 200 colonies per plate. The overlay is kept at 50°C and contains 2 x TY, 0.2% CMC, 0.75% agar and 100 ⁇ g ampicillin per ml. Plates are covered with 5 ml 0.5% agarose, 0.2% CMC and 100 ⁇ g ampicillin per ml kept at 50°C.
  • Example IV.1 Screening for endo-xylanase activity.
  • the method is similar to that used for cellulase-producing colonies in Example III.
  • the bottom layer contains 2 x TY, 1 .5% agar and 100 ⁇ g ampicillin per ml.
  • Cells are plated out in an overlay containing 2 x TY, 0.2 % oat spelt xylan (Sigma X-0627) 0.75 % agar and 100 ⁇ g ampicillin per ml.
  • the top layer contains 0.5% agarose, 0.1 % RBB-xylan (Sigma M501 ) and 100 ⁇ g ampicillin per ml in 25 mM phosphate buffer pH7.4.
  • the plasmid library was also plated on minimal medium plates containing 2% oat spelt xylan. After two days at 37°C some colonies gave halos, indicating the production of endo-xylanase. Other colonies gave rise to precipitation rings around the colonies. Five of the latter colonies were partially sequenced. They were similar to the arabinoxylan degrading cDNA whose isolation is described in pending application EP 94202442.3. A colony containing an A. niger axdA cDNA was deposited at the CBS on 3 August 1995 under CBS 592.95. The DNA sequence of the insert is as shown in SEQ ID No. 9, together with the amino acid sequence it encodes.
  • ORGANISM Aspergillus niger
  • ORGANISM Aspergillus niger
  • GGT GCA AGC CAG TAC ATT TTC GTC GAA
  • GGC AAC TCC TGG ACC GGA GCT 628
  • Gly Ala Ser Gin Tyr lie Phe Val Glu Gly Asn Ser Trp Thr Gly Ala 185 190 195
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus niger
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus niger
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • ORGANISM Aspergillus niger
  • GGC ATA TAC CTC ATT GCA TTG GCC CCC TTT GTC AAC GCA AAA TGC
  • GCT 158 Gly Ile Tyr Leu Ile Ala Leu Ala Pro Phe Val Asn Ala Lys Cys Ala 15 20 25
  • MOLECULE TYPE protein

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Cette invention concerne un procédé d'identification d'un fragment d'ADN codant une protéine à analyser. D'après ce procédé, on crible une banque d'ADNc, se présentant sous forme de cellules hôtes bactériennes transformées par de l'ADN obtenu dans un organisme eucaryote capable de produire ladite protéine, ceci afin de déceler l'expression de ladite protéine à l'aide d'un test qui permet de révéler sa présence. Ce procédé se caractérise par le fait que le criblage des cellules hôtes se fait une fois que l'ADN de transformation est devenu partie d'un plasmide. La banque d'ADNc provient de préférence d'un organisme eucaryote. Il est souhaitable que cet organisme eucaryote soit un champignon ou une cellule de levure, de préférence une espèce Aspergillus, et idéalement, un élément du groupe Aspergillus niger. Les enzymes ainsi criblés consistent, de préférence, en des enzymes présentant une activité de dégradation d'arabinoxylane, d'endoxylanase ou de cellulase.
PCT/EP1996/004510 1995-10-13 1996-10-14 Detection de proteines WO1997013853A2 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU72943/96A AU7294396A (en) 1995-10-13 1996-10-14 Protein detection
EP96934722A EP0796328A2 (fr) 1995-10-13 1996-10-14 Detection de proteines
JP9514730A JPH10510720A (ja) 1995-10-13 1996-10-14 タンパク質検出法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP95202777 1995-10-13
EP95202777.9 1995-10-13

Publications (2)

Publication Number Publication Date
WO1997013853A2 true WO1997013853A2 (fr) 1997-04-17
WO1997013853A3 WO1997013853A3 (fr) 1997-06-19

Family

ID=8220719

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1996/004510 WO1997013853A2 (fr) 1995-10-13 1996-10-14 Detection de proteines

Country Status (4)

Country Link
EP (1) EP0796328A2 (fr)
JP (1) JPH10510720A (fr)
AU (1) AU7294396A (fr)
WO (1) WO1997013853A2 (fr)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2786784A1 (fr) * 1998-12-04 2000-06-09 Lesaffre & Cie FRAGMENT D'ADN CODANT POUR LA XYLANASE THERMOSTABLE XynA DE THERMOASCUS
WO2001079507A3 (fr) * 2000-04-13 2002-02-07 Mark Aaron Emalfarb Nouvelles sequences de regulation de l'expression et produits d'expression dans le domaine des champignons filamenteux
US6573086B1 (en) 1998-10-06 2003-06-03 Dyadic International, Inc. Transformation system in the field of filamentous fungal hosts
US7220542B2 (en) 2000-07-17 2007-05-22 Van Den Brink Johannes Maarten Expression cloning in filamentous fungi
EP1862540A1 (fr) * 2003-05-29 2007-12-05 Genencor International, Inc. Nouveaux gènes de Trichoderma
WO2008037757A1 (fr) 2006-09-29 2008-04-03 Novozymes A/S Xylanases pour aliments pour animaux
US7374925B2 (en) 1998-05-06 2008-05-20 Adisseo France Sas Penicillium funiculosum mutant strain
WO2010121933A1 (fr) * 2009-04-22 2010-10-28 Dsm Ip Assets B.V. Procédé de production d'un polypeptide recombinant d'intérêt
EP2319920A1 (fr) 2005-12-22 2011-05-11 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
US8043839B2 (en) 2006-02-14 2011-10-25 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
WO2013037933A3 (fr) * 2011-09-14 2013-05-30 Dupont Nutrition Biosciences Aps Enzymes
US8486680B2 (en) 2007-10-03 2013-07-16 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
US8728769B2 (en) 2002-06-14 2014-05-20 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
EP2678426A4 (fr) * 2011-02-25 2015-03-25 Univ Saskatchewan Protéines pour la lutte biologique contre des champignons pathogènes de plantes
US9012186B2 (en) 2009-04-27 2015-04-21 The Board Of Trustees Of The University Of Illinois Hemicellulose-degrading enzymes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ239085A (en) * 1990-07-24 1993-08-26 Gist Brocades Nv Cloning and expression of xylanase genes from fungal origin
US5863783A (en) * 1991-03-27 1999-01-26 Gist-Brocades, N.V. Cloning and expression of DNA molecules encoding arabinan-degrading enzymes of fungal origin
MX9206979A (es) * 1991-12-04 1993-07-01 Novo Nordisk As Metodo de clonacion de proteinas en levaduras

Cited By (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8043844B2 (en) 1998-05-06 2011-10-25 Adisseo France Sas Mixture obtained from penicillium funiculosum
US7374925B2 (en) 1998-05-06 2008-05-20 Adisseo France Sas Penicillium funiculosum mutant strain
US6573086B1 (en) 1998-10-06 2003-06-03 Dyadic International, Inc. Transformation system in the field of filamentous fungal hosts
US7399627B2 (en) 1998-10-06 2008-07-15 Dyadic International (Usa), Inc. Transformation system in the field of filamentous fungal hosts
US7906309B2 (en) 1998-10-06 2011-03-15 Dyadic International (Usa), Inc. Expression-regulating sequences and expression products in the field of filamentous fungi
FR2786784A1 (fr) * 1998-12-04 2000-06-09 Lesaffre & Cie FRAGMENT D'ADN CODANT POUR LA XYLANASE THERMOSTABLE XynA DE THERMOASCUS
US8067157B2 (en) 1998-12-22 2011-11-29 Dsm Ip Assets B.V. Expression cloning in filamentous fungi
WO2001079507A3 (fr) * 2000-04-13 2002-02-07 Mark Aaron Emalfarb Nouvelles sequences de regulation de l'expression et produits d'expression dans le domaine des champignons filamenteux
US7220542B2 (en) 2000-07-17 2007-05-22 Van Den Brink Johannes Maarten Expression cloning in filamentous fungi
US9765319B2 (en) 2002-06-14 2017-09-19 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
US8728769B2 (en) 2002-06-14 2014-05-20 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
US8202704B2 (en) 2003-05-29 2012-06-19 Danisco Us Inc. Trichoderma genes
EP1862540A1 (fr) * 2003-05-29 2007-12-05 Genencor International, Inc. Nouveaux gènes de Trichoderma
US7923235B2 (en) 2003-05-29 2011-04-12 Danisco Us Inc. CIP1 polypeptides and their uses
US7666648B2 (en) 2003-05-29 2010-02-23 Danisco Us Inc. Isolated polypeptide having arabinofuranosidase activity
US9593324B2 (en) 2005-12-22 2017-03-14 Roal Oy Treatment of cellulosic material and enzymes useful therein
EP2453014A1 (fr) 2005-12-22 2012-05-16 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
EP2453013A1 (fr) 2005-12-22 2012-05-16 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
EP2319920A1 (fr) 2005-12-22 2011-05-11 ROAL Oy Traitement de matériel cellulosique et enzymes pouvant être employées dans ce traitement
US8409836B2 (en) 2005-12-22 2013-04-02 Roal Oy Treatment of cellulosic material and enzymes useful therein
US9758777B2 (en) 2005-12-22 2017-09-12 Roal Oy Treatment of cellulosic material and enzymes useful therein
USRE45660E1 (en) 2006-02-14 2015-09-01 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
US8043839B2 (en) 2006-02-14 2011-10-25 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
WO2008037757A1 (fr) 2006-09-29 2008-04-03 Novozymes A/S Xylanases pour aliments pour animaux
US8486680B2 (en) 2007-10-03 2013-07-16 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
USRE46733E1 (en) 2007-10-03 2018-02-27 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
WO2010121933A1 (fr) * 2009-04-22 2010-10-28 Dsm Ip Assets B.V. Procédé de production d'un polypeptide recombinant d'intérêt
US9012186B2 (en) 2009-04-27 2015-04-21 The Board Of Trustees Of The University Of Illinois Hemicellulose-degrading enzymes
EP2678426A4 (fr) * 2011-02-25 2015-03-25 Univ Saskatchewan Protéines pour la lutte biologique contre des champignons pathogènes de plantes
EP3061816A1 (fr) * 2011-09-14 2016-08-31 DuPont Nutrition Biosciences ApS Utilisation d'enzymes avec activité endo-1,4-xylanase pour réduire du mauvais gout dans des produits
US9683224B2 (en) 2011-09-14 2017-06-20 Dupont Nutrition Biosciences Aps Enzymes
EA027084B1 (ru) * 2011-09-14 2017-06-30 ДюПон НЬЮТРИШН БАЙОСАЙЕНСИЗ АпС Ферменты
CN103814129A (zh) * 2011-09-14 2014-05-21 杜邦营养生物科学有限公司 包含具有内切–1,4–β–木聚糖酶活性的酶和具有内切–1,3(4)–β葡聚糖酶活性的酶的组合物
WO2013037933A3 (fr) * 2011-09-14 2013-05-30 Dupont Nutrition Biosciences Aps Enzymes
AU2012307299B2 (en) * 2011-09-14 2018-03-22 International N&H Denmark Aps Compositions comprising enzymes with endo - 1, 4 - beta - xylanase activity and enzymes with endo - 1, 3 (4) - beta glucanase activity
AU2012307299C1 (en) * 2011-09-14 2018-10-25 International N&H Denmark Aps Compositions comprising enzymes with endo - 1, 4 - beta - xylanase activity and enzymes with endo - 1, 3 (4) - beta glucanase activity

Also Published As

Publication number Publication date
AU7294396A (en) 1997-04-30
JPH10510720A (ja) 1998-10-20
EP0796328A2 (fr) 1997-09-24
WO1997013853A3 (fr) 1997-06-19

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