Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-03-14
2004-10-19
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S254110, C435S254300, C435S320100, C435S069100, C536S023740
Reexamination Certificate
active
06806062
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to isolated nucleic acid sequences encoding polypeptides having transcriptional activation activity and to the polypeptides. The invention also relates to nucleic acid constructs, vectors and host cells comprising the nucleic acid sequences. The invention further relates to host cells useful for the production of polypeptides in which the production or function of the transcriptional activator has been altered, as well as to methods for producing the polypeptides.
2. Description of the Related Art
The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including eubacterial and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce adequate yields of the protein in an active state, but, to a large extent, may also be governed by the intended end use of the protein. One problem frequently encountered is the high level of proteolytic enzymes produced by a given host cell or present in the culture medium. One suggestion has been to provide host organisms deprived of the ability to produce specific proteolytic compounds. For example, WO 90/00192 (Genencor, Inc.) describes filamentous fungal hosts incapable of secreting enzymatically active aspartic proteinase. EP 574 347 (Ciba Geigy AG) describes Aspergillus hosts defective in a serine protease of the subtilisin-type. WO 98/12300 (Novo Nordisk A/S) describes hosts defective in a metalloprotease and an alkaline protease. WO 97/12045 (Genencor, Inc.) describes yeast and bacterial host systems, which are rendered protease deficient resulting from a disruption of a promoter sequence involved in the regulation of a protease gene.
Mattern, I. E., et al., (1992. Mol Gen Genet 234:332-336) describe a mutant strain of
Aspergillus niger
, which was shown to have only 1 to 2% of the extracellular protease activity of the parent strain, apparently due to a deficiency of at least two proteases, aspergillopepsin A and aspergillopepsin B. It was suggested that the protease deficient phenotype could result from a regulatory mutation affecting the expression of the genes coding for both proteases.
The initiation of eukaryotic transcription at a specific promoter or set of promoters requires a eukaryotic transcriptional activator which is a polypeptide, but which is not itelf part of RNA polymerase. Many transcriptional activators bind to a specific site on the promoter to form a functional promoter necessary for the initiation of transcription of the polypeptide encoding sequence. However, a transcriptional activator may also be incorporated into an initiation complex only in the presence of other polypeptides. Polypeptides with transcriptional activation activity have been described in fungi, and a list of such polypeptides has been published (Dhawale, S. S., and Lane, A. C. 1993. Nucleic Acid Research 21:5537-5546).
Solution Proposed by the Invention:
It is an object of the present invention to provide improved methods for increasing production of polypeptides in host cells in which the activity of a transcriptional activator involved in the regulation of protease production has been modified.
SUMMARY OF THE INVENTION
A first aspect of the present invention relates to an isolated nucleic acid sequence encoding a transcriptional activator selected from the group consisting of:
(a) a nucleic acid sequence having at least 70% identity with the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO: 48;
(b) a nucleic acid sequence encoding a polypeptide having an amino acid sequence which has at least 50% identity with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 49;
(c) a nucleic acid sequence which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO: 48, or (ii) its complementary strand, wherein the low stringency conditions are defined by prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 microg/ml sheared and denatured salmon sperm DNA, and 25% formamide, and wash conditions are defined by 50° C. for 30 minutes in 2×SSC, 0.2% SDS;
(d) an allelic variant of (a), (b), or (c);
(e) a subsequence of (a), (b), (c), or (d), wherein the subsequence encodes a polypeptide fragment which has transcriptional activation activity; and
(f) a subsequence of (a), (b) (c), or (d), wherein the subsequence encodes a polypeptide with the amino acid sequence of SEQ ID NO:3.
The nucleic acid sequence shown in SEQ ID NO: 1 is the
Aspergillus niger
prtT gene encoding the transcriptional factor shown in SEQ ID NO: 2 as described further below and in the Examples.
The nucleic acid sequence shown in SEQ ID NO: 48 is the
Aspergillus oryzae
IFO4177 prtT gene encoding the transcriptional factor shown in SEQ ID NO: 49. The
A. oryzae
prtT gene has a coding region starting in position 795 and ending at position 2931. The prtT gene has 4 introns in positions 1028-1135, 1538-1591, 2018-2066, and 2297-2347, respectively. This is described further below and in the Examples.
In another aspect, the invention also relates to nucleic acid constructs, vectors and host cells comprising the nucleic acid sequences, and to the polypeptides encoded by the nucleic acid sequences. The invention further relates to host cells useful for the production of a polypeptide, in which the production or function of the transcriptional activator has been altered, as well as to methods for producing the polypeptide.
REFERENCES:
patent: 6007988 (1999-12-01), Choo et al.
patent: 0 574 347 (1993-12-01), None
patent: WO 90/00192 (1990-01-01), None
patent: WO 95/35385 (1995-12-01), None
patent: WO 97/12045 (1997-04-01), None
patent: WO 97/35956 (1997-10-01), None
patent: WO 98/12300 (1998-03-01), None
Mattern et al (1992) Mol. Gen. Genet. 234:332-336.*
Broun et al (1998) Science 282:1316-1318.*
Dhawale et al., Oxford University Press, Nucleic Acids Research, 21(24):5537-5546 (1993).
Mattern et al., Mol. Gen. Genet, 234:332-336 (1992).
Swiss-Prot database, acc. No. P38114, Entian et al., pp. 106-142 (Oct. 1, 1994).
Swiss-Prot database, acc. No. P39529, Purnelle et al., 10:1235-1249 (1994) Yeast.
Swiss-Prot database, accession No. P28348, Burger et al., 11:5746-5755 (1991) Mol. Cell. Biol.
Swiss-Prot database, accession No. P87000, Bibbins et al. (Nov. 1, 1997).
van den Hombergh et al., (1997) Curr. Genet. 32:73-81.
van den Hombergh et al., (1997) Tibtech 15:256-263.
Christensen Tove
Hjort Carsten M.
Punt Peter J.
Schuren Frank H. J.
van den Hondel Cees
Ketter James
Lambiris Elias J.
Novozymes A/S
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