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
1998-09-04
2001-09-18
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...
C435S484000, C435S254300, C435S320100
Reexamination Certificate
active
06291209
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel fungal host cells and to methods of producing proteins. More specifically, the invention relates to a host cell useful for the expression of heterologous proteins, in which the host cell has been genetically modified in order to express significantly reduced levels of alkaline protease activity. Moreover, the invention relates to a method of producing proteins of interest in high yields by using the fungi of the invention, in which the method comprises cultivating the host cell in a suitable growth medium, followed by recovery of the desired protein. The invention also comprises methods for producing such fungi and DNA constructs to be used in these methods.
BACKGROUND OF THE INVENTION
Fungi, and especially filamentous fungi, are widely used commercially as host cells for the expression of proteins which are secreted extracellularly. In particular, species belonging to the genus Aspergillus have a long history of commercial use for the production of endogenous and lately also heterologous proteins.
One disadvantage frequently encountered with microorganisms used as host cells is the inherent production and secretion of high levels of proteolytic enzymes which may result in reduced yields of a protein product of interest due to proteolysis.
Various solutions to circumvent this have been envisaged. For example, one could delete or disrupt the genes encoding the various endogenous proteases. WO 90/00192 (Genencor Inc.) describes mutant filamentous fungal hosts which have been rendered incapable of secreting enzymatically active aspartic protease. By such mutation, it was shown that the yield of the heterologous polypeptide, bovine chymosin, was increased. EP 574 347 (Ciba Geigy AG) describes an Aspergillus host defective in a serine protease of the subtilisin type.
However, it is well known that fungi produce a large number of proteases in addition to the two herein mentioned. Consequently, strains of filamentous fungi exhibiting no or very low levels of proteolytic activity originating from other proteases are still needed.
SUMMARY OF THE INVENTION
It is an objective of the present invention to provide filamentous fungal host cells for the production of heterologous polypeptide products in which the cell has been genetically modified in order to express significantly reduced levels of an endogenous alkaline protease, by comparison to the parental cell.
The present invention relates to such modified fungi, wherein transcription and/or translation of at least one gene encoding an alkaline protease activity has been completely or partially inhibited.
According to the invention, this may be accomplished through the use of recombinant DNA technology wherein gene sequences encoding alkaline protease activity of the fungus in question is deleted or mutated, resulting in reduced levels of protease expression or expression of a protease with reduced levels of activity.
In a specific embodiment the alkaline protease gene is the alp gene shown in SEQ ID No 1 and described by Murakami, K., et al., 1991, Agric. Biol. Chem. 55:2807-2811.
Furthermore, the invention relates to methods for producing such fungi, wherein the inactivation of the alkaline gene(s) is(are) obtained by nucleotide substitution, deletion or insertion in the alkaline protease gene.
The invention also relates to processes for producing heterologous polypeptides using fungal host cells of the invention. Especially contemplated are secreted polypeptides, wherein a fungal host cell, modified and optionally transformed with a DNA construct comprising at least a DNA sequence encoding the polypeptide or gene product of interest, is cultivated in a suitable growth medium under appropriate conditions and the desired gene product is recovered and purified.
By the methods described herein, it has been found that the yield of polypeptides secreted by fungi of the invention is much improved.
Furthermore, the invention relates to polypeptide products produced by the above methods.
Lastly, the invention relates to DNA constructs comprising the DNA sequence of the alp gene of
Aspergillus oryzae
intended for use in the above mentioned methods.
REFERENCES:
patent: 0 414 297 A1 (1981-02-01), None
patent: 0 574 347 A2 (1993-12-01), None
patent: WO 90/00192 (1990-01-01), None
patent: WO 92/17595 (1992-10-01), None
Murakami et al., Agric. Biol. Chem., vol. 55, 1991, pp. 2807-2811.*
Dialog Information Service, File 155, Medline, Accession No. 7701294.
Dialog Information Service, File 5, Biosis, Accession No. 11494130.
Dialog Information Service, File 155, Medline, Accession No. 07408772.
Tang et al., “AnAspergillus FumigatusAlkaline Protease Mutant Constructed By Gene Disruption Is Deficient In Edtracellular Elastase Activity”, Molecular Mocrobiology (1992) 6 (12), pp. 1663-1671.
Tatsumi et al., “A Full Length cDNA Clone For The Alkaline Protease FromAsperfillus Oryzaie: Structural Analysis And Expression inSaccharomyces Cerevisiae”, Mol. Gen. Genet (1989) 219, pp. 33-38.
Gorbell Jason I.
Ketter James
Novo Nordisk A/S Novo Alle
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