WO2001085925A3

WO2001085925A3 - Verfahren zum herstellen von proteinen in einer hefe der gattung arxula und dafür geeignete promotoren

Info

Publication number: WO2001085925A3
Application number: PCT/EP2001/005225
Authority: WO
Inventors: Gotthard Kunze; Ines Walter; Harald Roesel; Erik Boeer; Regina Stoltenburg; Thomas Wartmann; Bui Minh Duc
Original assignee: Institut fuer Pflanzengenetik und Kulturpflanzenforschung; Rhein Biotech GmbH
Current assignee: Institut fuer Pflanzengenetik und Kulturpflanzenforschung; Dynavax GmbH
Priority date: 2000-05-08
Filing date: 2001-05-08
Publication date: 2002-07-11
Anticipated expiration: 2002-11-08
Also published as: DE50115564D1; EP1280893B1; US20030186376A1; AU2001263920A1; WO2001085925A2; DE10022334A1; ATE474928T1; EP1280893A2

Abstract

Die Erfindung betrifft ein Verfahren zum Herstellen von einem oder mehreren Proteinen in einer Wirtszelle der Hefegattung Arxula und ein Nukleinsäure-Molekül, das einen dafür geeigneten Promotor umfasst. Ferner betrifft die vorliegende Erfindung einen Expressionsvektor, eine Wirtszelle und einen Kit sowie die Verwendung derselben. Aufgabe der Erfindung ist es, ein Verfahren zum Herstellen eines Proteins in einer Hefe der Gattung Arxula bereitzustellen, mit dem höhere Proteinmengen erhalten werden können. Diese Aufgabe wird gelöst durch ein Verfahren zum Herstellen von einem oder mehreren Proteinen in einer Wirtszelle der Hefegattung Arxula, umfassend: (i) Klonieren von mindestens einer Nukleinsäure, die ein heterologes Protein kodiert, in einen Expressionsvektor, der einen induzierbaren Promotor aus einer Hefe der Gattung Arxula oder einen Promotor von einem konstitutiv exprimierten Gen, ausgewählt aus dem ARFC3-Gen und dem AHSB4-Gen, aus einer Hefe der Gattung Arxula enthält, so dass die klonierte Nukleinsäure unter der Transkriptionskontrolle des Promotors steht; (ii) Einführen des in (i) erhaltenen Expressionsvektors in eine zum Herstellen des heterologen Proteins geeignete Wirtszelle der Hefegattung Arxula; (iii) Kultivieren der in (ii) erhaltenen Wirtszelle und gegebenenfalls (iv) Induzieren des Promotors; (v) Ernten des Proteins in an sich bekannter Weise.

Description

A CLASSIFICATION OF SUBJECT MATTER

IPC 7 C12N15/81 //C12R1/645

International Patent Classification (IPC) or to both national classification and IPC

B FIELDS SEARCHED

Minimum documentation searched (classification System followed by classification Symbols) IPC 7 C12N

Documentation searched other than minimum documentation to the extent that such documents are included in the fϊelds searched

Electronic data base consulted durmg the international search (name of data base and, where practical, search terms used) EPO-Internal, WPI Data, PAJ, BIOSIS, CHEM ABS Data, EMBASE

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No

X DE 44 25 058 A (INST PFLANZENGENETIK & 1,4-6, KULTUR) 18 January 1996 (1996-01-18) 25,26, 29-31, 33-35, 47-49 52-59

Figure 1 Example 4 1.

X DD 298 821 A (ZI FUER GENETIK UND 1,2,4-6, KULTURPFLA) 12 March 1992 (1992-03-12) 25,26,

29,30

Abstract

Further documents are hsted in the contmuation of box C ^Patent family members are hsted in annex

* Special categoπes of cited documents "T" later document pubhshed after the international fihng date or pπoπty date and not in conflict with the apphcation but cited to

"A" document definmg the general State of the art which is not understand the pπnciple or theory underlymg the invention considered to be of particular relevance

"X" document of particular relevance, the claimed invention cannot be

"E" earher document but pubhshed on or after the international fihng considered novel or cannot be considered to mvolve an inventive date step when the document is taken alone

"L" document which may throw doubts on pπoπty claιm(s) or which "Y" document of particular relevance, the claimed invention cannot be is cited to estabhsh the pubheation date of another citation or considered to mvolve an inventive step when the document is other Special reason (as specifϊed) combmed with one or more other such documents, such combmation being obvious to a person skilled in the art

"O" document referπng to an oral disclosure, use, exhibition or other means "&" document member of the same patent family

"P" document pubhshed pπor to the international fϊling date but later than the pπoπty date claimed

Date of the actual completion of the international search Date of maihng of the international search report 11 December 2001 (11 12 2001) 18 January 2002 (18.01.2002)

Name and maihng address of the Authoπzed officer EUROPEAN PATENT OFFICE

Telephone No.

Form PCT/ISA/210 (second sheet) (July 1992) l of 3

. PCT/EP 01/05225

C. (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citaüon of document, with indication, where appropπate, of the relevant passages Relevant to claim No.

WARTMANN T ET AL: "AIL VI GENE FROM THE 1-12,

YEAST ARXULA ADENINIVORANS LS3 - A NEW 25-36,

SELECTIVE TRANSFORMATION MARKER" 38-40,

YEAST, CHICHESTER, SUSSEX. GB, 44-59

Bd. 14 August 1998 (1998-08), Seiten

1017-1025, XP000985335

ISSN: 0749-503X page 1022 page 1024

INTERNATIONAL SEARCH REPORT [nteπ-auon-u appticauon No.

PCT/EPO 1/05225

Box i Observanons where certain Claims were found utuearchable (Continuation of item 1 of first sheet)

This internauotiai searchrepoπ has not beert established inrespeα of ceπatn ciaims under Aiticie 17(2)(a) forthe followingrensoos-

M Claims Nos.:

^^ because they reiate to subiect matter not required to be searched by this Authoπty, nameiy:

In so far as an "in vivo" method is concerned, claim 58 refers to a method for the treatment of the human/animal body, the research was carried out and based on the given effects of the compound/composition.

| [ ClaimsNos.: because thev reiate to paπs ot^' the international apphcauon that do not compiy with the prescπbed requirements to such an extent that no meaningM intemationai search can be carned out. speciπcaliy:

D Claims N^'os.: because thev are αependem clauns and are not dratted in accordance with the second and third semences of Rule 6.4(a).

Box II Observanons where unity of invention is lac ing tContinuation of item 2 of first sheet)

This Intemauonai Searching Authoπry found muiupie ιnvemιons in this interπationai appiication, as follows:

See supplemental sheet

As a result of the provisional examination according to PCT Article 40.2(e) no additionai fees are to be refunded.

1 I I As all required additionai searcn tees vvere tunetv paid bv the appiicant. this intemauonai search repoπ Covers aü - arcnable Claims.

2. I I As all searchable clauns could be searcned withcut etfoπ justiry-ng an additionai fee. this Authoπrv did not inv-tepayment oi any additionai fee.

As oniv some of the requireα additionai search fees were tuneiv paid bv the appiicant. this intemationai search repoπ covers oniy those ciaims ior wruch fees were paid. speciήcailv clauns Nos.:

(2-15, 31-36, 41, 43) fully, (1, 25-30, 38-40, 44-59) partly

G No requireα additionai search teεs ere timeiv paid bv the aDpiicant. Consequeniiv. this - emationai search repoπ is restπcteα to the invention rirst meniioned in ine claims: u is covereα by Claims Nos.

Remark on Protest ^ \ The aαditionai searcn fees were accompamed bv the appiicant s protest.

I No protest accompanieα the payment of addiüonal search fees.

Form PCT ISA/210 (coMimiai-on of first sheet ( 1 )) (July 1 92) n ona app ca on o.

PCT/EP01/05225

Correspondmg to invention 2 but referring to the promoters with SEQ. ID Nos: 7, 8 or 9.

5. Claims: (10-12, 36,) fully, (1, 25-30, 38-40, 47-59) partly

Correspondmg to invention 2 but referring to the promoters with SEQ. ID Nos: 10 or 11.

6. Claims: (13-15, 41) fully, (1, 25-30, 44-59) partly

Correspondmg to invention 2 but referring to the promoter with SEQ. ID No: 12.

7. Claims: (16-21, 37) fully, (1, 25-30, 38-40, 47-59) partly

Corresponding to invention 2 but referring to the promoters with SEQ. ID Nos: 13 or 14.

8. Claims: (22-24, 42) fully, (1, 25-30, 44-59) partly

Corresponding to invention 2 but referring to the promoter with SEQ. ID No: 18.

Form PCT ISA 210 INTERNATIONALSEARCH REPORT Intemationai apphcation No. PCT/ΕPO 1/05225

ADDITIONAL MATTER PCT/ISA 210

The international search authority has established that this international apphcation contains multiple inventions, as follows:

1. Claims: 2 fully, (1, 25-30) partly

Method for the production of one or several proteins in a host cell of the yeast genus Arxula, comprising: cloning at least one nucleic acid coding for a heterologous protein in an expression vector, containing ARFC3 -promoter SEQ. ID NO:l or 2 ( and which can furthermore contain marker genes containing SEQ. ID NO: 15 or 16 and/or the sequences permitting Integration in the genome of the host cell and or the sequences permitting replication in the host cell), such that the cloned nucleic acid is under the transcription control of the said promoter, introduction of the above vector into a host cell of the yeast genus Arxula, suitable for the production of the heterologous protein, cultivation of the host cell, optional induction of the promoter and harvesting the protein.

2. Claims: (3, 43) fully, (1, 25-30, 44-59) partly

Method for the production of one or several proteins in a host cell of the yeast genus Arxula, comprising: cloning at least one nucleic acid coding for a heterologous protein in an expression vector, containing AHSB4-promoter SEQ. ID NO: 17 ( and which can furthermore contain marker genes containing SEQ. ID NO: 15 or 16 and/or the sequences permitting Integration in the genome of the host cell and/or the sequences permitting replication in the host cell), such that the cloned nucleic acid is under the transcription control of the said promoter, introduction of the above vector into a host cell of the yeast genus Arxula, suitable for the production of the heterologous protein, cultivation of the host cell, optional induction of the promoter and harvesting the protein.

Nucleic acid molecules comprising promoter SEQ. ID NO: 17 and derivatives or fragments of said sequence, an expression vector containing said nucleic acid molecule which can furthermore contain marker genes containing SEQ. ID NO: 15 or 16 and/or the sequences permitting integration in the genome of a host cell of the yeast genus Arxula and/or the sequences permitting replication in the host cell, host cells containing said vector and/or the above nucleic acid molecule, use of the nucleic acid molecule, the expression vector or the host cell for the production of one or more proteins.

3. Claims: (4-6, 31) fully, (1, 25-30, 33-35, 47-59) partly

Corresponding to invention 2 but referring to the promoters with SEQ. ID Nos: 3, 4, 5, or 6.

4. Claims: (47-9, 32) fully, (1, 25-30, 33-35, 47-59) partly

CT ISA 210 INTERNATIONAL SEARCH REPORT International Apphcation No lnformauon on patent tamily members PCI/EP 01/05225

Patent document Publicauon Patent famdiy Publicauon cited in search repoπ date member(s) date

DE 4425058 A 18-01-1996 DE 4425058 AI 18-01-1996

DD 298821 A 12-03-1992 DD 298821 A5 12-03-1992