Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1997-08-27
1999-01-19
Ketter, James
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
43525411, C12P 2102
Patent
active
058612807
DESCRIPTION:
BRIEF SUMMARY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/DK96/29391 filed 20 Mar. 1996 and claims priority under 35 U.S.C. 119 of Danish application 0284/95 filed 20 Mar. 1995, the contents of which are fully incorporated herein by reference.
TECHNICAL FIELD
The present invention relates to novel host cells and to methods of producing proteins. More specifically the invention relates to a host cell useful for the expression of heterologous proteins, which host cell has been genetically modified in order to express significantly reduced levels of a metalloprotease. Moreover the invention relates to a method of producing a heterologous protein, which method comprises cultivating the host cell in a suitable growth medium, followed by recovery of the desired protein.
BACKGROUND 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 eucaryotic hosts. The selection of an appropriate expression system often not only depends on the ability of the host cell to produce adequate yields of the protein in an active state, but also to a large extent may 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 in the culture medium. It has been suggested that one could provide host organism deprived of the ability of producing specific proteolytic compounds. For example, International Patent Application WO 90/00192 describes filamentous fungal hosts incapable of excreting enzymatically active aspartic proteinase, and EP 574 347 describes Aspergillus hosts defective in a serine protease of the subtilisin-type.
Metalloproteases have been isolated from a number of eucaryotic sources. Neutral metalloproteases, i.e. metalloproteases having optimal activity at neutral pH, isolated from strains of Aspergillus also have been reported. Neutral metalloproteases have been classified into two groups, NpI and sequence of a neutral metalloprotease II cDNA from Aspergillus oryzae have Masaki A, Kawabe H, Arimura H, Nakano E and Motai H; Mol. Gen. Genet. 1991 228 97-103!. The nucleotide sequence of a neutral metalloprotease I cDNA from Aspergillus oryzae have never been disclosed.
Although metalloproteases have been reported, their role in relation to reducing the stability of the products obtained from these organisms have never been described.
SUMMARY OF THE INVENTION
According to the present invention it has now been found that metalloproteases may reduce significantly the stability of the product obtained by a cell.
Accordingly, the present invention provides a host cell useful for the expression of a heterologous protein product, which cell has been genetically modified in order to express significantly reduced levels of a metalloprotease, as compared to the parental cell.
In another aspect, the invention provides a method of producing a heterologous protein product in a host cell of the invention, which method comprises introducing into the host cell a nucleic acid sequence encoding the protein, cultivating the host cell in a suitable growth medium, and isolating the heterologous protein product.
By the method of the invention, the proteolytic action arising from metalloproteases have been significantly reduced, thereby improving the stability of the protein obtained by the method. Moreover, the protein obtained by the method of the invention can be obtained as a precursor protein, i.e. a zymogen, a hybrid protein, a protein obtained as a pro sequence or pre-pro sequence, or in unmaturated form.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention is further illustrated by reference to the accompanying drawing,
REFERENCES:
Tatsumi et al., "Cloning And Expression In Yeast Of A cDNA Clone Encoding Aspergillus Oryzae Neutral Protease II, A Unique Metalloprotease", Mol Gen Genet, 1991, 228: pp. 97-103.
Gregg, Esq. Valeta
Ketter James
Novo Nordisk A S
Zelson Esq. Steve T.
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