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
1998-03-26
2003-01-21
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...
C435S069400, C435S069510, C435S069600, C435S202000, C435S205000, C435S212000
Reexamination Certificate
active
06509171
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the production of polypeptides in suitable novel hosts. More particularly this invention relates to the production of heterologous polypeptides in novel filamentous fungal hosts which are incapable of excreting enzymatically active aspartic proteinase.
BACKGROUND OF THE INVENTION
The aspergillopepsins are a family of closely related aspartic proteinases produced by certain filaments fungi of the genus Aspergillus. They share extensive amino acid sequence homology with the penicillopepsins, which are aspartic proteinases produced by certain Penicillium species, (In Kosta, V. (Ed.), “Aspartic Proteinases and Their Inhibitors”, Walter de Gruyter, New York, pp.27-40, 1985). The aspergillopepsins also share regions of homology with aspartic proteinases from other filamentous fungi such as
Mucor miehei
(
Neth. Milk Dairy J.,
35, pp. 275-280, 1981),
Rhizopus chinensis
(
Can. J. Biochem.,
51, pp. 789-796, 1973), and
Endothia parasitica,
(
Eur. J. Biochem.,
167, pp.327-338, 1987). The degree of sequence conservation appears to be greatest in the regions surrounding active site amino acid residues. The complete amino acid sequence of aspergillopepsin A from
A. awamori
has been reported (
Bioorg. Khim.,
8, pp. 1030-1047, 1986). The mature enzyme is composed of a single polypeptide chain of 328 amino acids. Genes encoding the aspartic proteinases of
Mucor miehei
(
Gene,
48, pp.41-53, 1986;
Proteins
1, pp. 363-369, 1986), and
Rhizopus chinensis
(
J. Biol. Chem.,
262, pp. 1461-1467, 1987), have been cloned and their nucleotide sequences have provided information that these enzymes are synthesized as zymogen precursors. Fungal aspartic proteinases have been studied intensely and considerable information is available regarding the structure-function relationships (
Biochim. Biophys. Acta,
336, pp. 437-444, 1974), as well as the three-dimensional structures of some of these enzymes (
Nature,
267, pp. 808-813, 1977;
Nature,
266, pp. 140-145, 1977;
J. Mol. Biol.,
196, pp. 877-900,
FEBS Lett.,
174, pp. 96-101, 1984; “Aspartic Proteinases and Their Inhibitors”, Walter de Gruyter, New York, pp. 151-161 and 163-177, 1985).
Genomic DNA sequences encoding the
Mucor miehei
aspartic proteinase gene were isolated by Gray et al. (
Gene,
48, pp. 41-53, 1986). The nucleotide sequence of this gene indicated that it contained no intervening sequences.
Filamentous fungi have recently been used as hosts for the expression and secretion of heterologous protein products (
Bio/Technol.,
5 pp. 369-376, 1987;
Bio/Technol.,
5 pp. 713-719, 1987;
Bio/Techno.,
5, pp. 1301-1304, 1987). While such aspartic acid proteinases from filamentous fungal hosts might degrade a heterologous polypeptide if left in contact with it for sufficient time, in vitro rapid separation of the protein has been thought to be sufficient to prevent any interference of aspartic proteinase with expression of the heterologous polypeptide expressed in filamentous fungi.
SUMMARY OF THE INVENTION
It has been discovered that when a filamentous fungus has the gene sequence corresponding to the aspartic proteinase produced therefrom inactivated or eliminated entirely by site specific DNA deletion in the gene sequence coding for the aspartic proteinase that such a fungus when used as a host for production of a heterologous polypeptide will surprisingly increase the production of the heterologous polypeptide produced thereby.
Accordingly, a novel mutant filamentous fungus and fungus culture is provided which is suitable for the production of heterologous polypeptides which contains a nonrevertable site-selected deletion that results in the filamentous fungus being incapable of excreting enzymatically active aspartic proteinase.
A method for producing a heterologous polypeptide in a filamentous fungus is described which comprises culturing a filamentous fungus which is capable of expressing the heterologous polypeptide and which contains a nonrevertable site-selected deletion that results in the filamentous fungus being incapable of excreting enzymatically active aspartic proteinase until an amount of the heterologous polypeptide has accumulated in the culture broth and then recovering the polypeptide.
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Berka Randy M.
Hayenga Kirk J.
Lawlis Virgil B.
Ward Michael
Genencor International Inc.
Genencor International Inc.
Ketter James
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