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
1997-11-24
2001-01-09
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700, C435S069800, C435S071100, C435S183000, C435S205000, C435S223000, C435S225000, C435S254110, C435S254300, C435S254400, C435S254800, C435S320100, C536S023100, C536S023200, C536S023740
Reexamination Certificate
active
06171817
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to heterologous polypeptides expressed and secreted by filamentous fungi and to vectors and processes for expressing and secreting such polypeptides. More particularly, the invention discloses transformation vectors and processes using the same for expressing and secreting biologically active bovine chymosin and heterologous glucoamylase by a filamentous fungus.
BACKGROUND OF THE INVENTION
The expression of DNA sequences encoding heterologous polypeptides (i.e., polypeptides not normally expressed and secreted by a host organism) has advanced to a state of considerable sophistication. For example, it has been reported that various DNA sequences encoding pharmacologically desirable polypeptides [e.g., human growth hormone (1), human tissue plasminogen activator (2), various human interferons (6), urokinase (5), Factor VIII (4), and human serum albumin (3)] and industrially important enzymes [e.g., chymosin (7), alpha amylases (8), and alkaline proteases (9)] have been cloned and expressed in a number of different expression hosts. Such expression has been achieved by transforming prokaryotic organisms [e.g.,
E. coli
(10) or
B. subtilis
(11)] or eukaryotic organisms [e.g.,
Saccharomyces cerevisiae
(7),
Kluyveromyces lactis
(12) or Chinese Hamster Ovary cells (2)] with DNA sequences encoding the heterologous polypeptide.
Some polypeptides, when expressed in heterologous hosts, do not have the same level of biological activity as their naturally produced counterparts when expressed in various host organisms. For example, bovine chymosin has very low biological activity when expressed by
E. coli
(13) or
S. cerevisiae
(7). This reduced biological activity in
E. coli
is not due to the natural inability of
E. coli
to glycosylate the polypeptide since chymosin is not normally glycosylated (14). Such relative inactivity, both in
E. coli
and
S. cerevisiae
, however, appears to be primarily due to improper folding of the polypeptide chain as evidenced by the partial post expression activation of such expressed polypeptides by various procedures. In such procedures, expressed chymosin may be sequentially denatured and renatured in a number of ways to increase biological activity: e.g., treatment with urea (13), exposure to denaturing/renaturing pH (13) and denaturation and cleavage of disulfide bonds followed by renaturation and regeneration of covalent sulfur linkages (15). Such denaturation/renaturation procedures, however, are not highly efficient [e.g., 30% or less recovery of biological activity for rennin (13)], and add considerable time and expense in producing a biologically active polypeptide.
Other heterologous polypeptides are preferably expressed in higher eukaryotic hosts (e.g., mammalian cells). Such polypeptides are usually glycopolypeptides which require an expression host which can recognize and glycosylate certain amino acid sequences in the heterologous polypeptide. Such mammalian tissue culture systems, however, often do not secrete large amounts of heterologous polypeptides when compared with microbial systems. Moreover, such systems are technically difficult to maintain and consequently are expensive to operate.
Transformation and expression in a filamentous fungus involving complementation of aroD mutants of
N. crassa
lacking biosynthetic dehydroquinase has been reported (16). Since then, transformation based on complementation of glutamate dehydrogenase deficient
N. crassa
mutants has also been developed (17). In each case the dehydroquinase (ga2) and glutamate dehydrogenase (am) genes used for complementation were derived from
N. crassa
and therefore involved homologous expression. Other examples of homologous expression in filamentous fungi include the complementation of the auxotrophic markers trpC, (18) and argB (19) in
A. nidulans
and the transformation of
A. nidulans
to acetamide or acrylamide utilization by expression of the
A. nidulans
gene encoding acetamidase (20).
Expression of heterologous polypeptides in filamentous fungi has been limited to the transformation and expression of fungal and bacterial polypeptides. For example,
A. nidulans
, deficient in orotidine-5′-phosphate decarboxylase, has been transformed with a plasmid containing DNA sequences encoding the pyr4 gene derived from
N. crassa
(21,32).
A. niger
has also been transformed to utilize acetamide and acrylamide by expression of the gene encoding acetamidase derived from
A. nidulans
(22).
Examples of heterologous expression of bacterial polypeptides in filamentous fungi include the expression of a bacterial phosphotransferase in
N. crassa
(23)
Dictyostellium discoideum
(24) and
Cephalosporium acremonium
(25).
In each of these examples of homologous and heterologous fungal expression, the expressed polypeptides were maintained intracellularly in the filamentous fungi.
Accordingly, an object of the invention herein is to provide for the expression and secretion of heterologous polypeptides by and from filamentous fungi including vectors for transforming such fungi and processes for expressing and secreting such heterologous polypeptides.
SUMMARY OF THE INVENTION
The inventor includes novel vectors for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources.
The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3′ terminus of the DNA sequence encoding the heterologous polypeptides.
Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus.
REFERENCES:
patent: 4486533 (1984-12-01), Lambowitz
patent: 4666847 (1987-05-01), Alford et al.
patent: 4794175 (1988-12-01), Nunberg et al.
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Moir, et al., “Molecular Cloning and characterization of Double-Stranded cDNA coding for Bovine Chymisin,” Gene, 19:127-138 (1982).
Nishimori, et al., “Nucleotide Sequeence of Calf Proremmin cDNA Cloned inEscherichia coli,”J. Biochem., 91:1085-1088 (1982).
Hidaka, et al., “Cloning and Structural Analysis of the Calf Prochymosin Gene,” Gene, 43:197-203 (1986).
Heyneker, et al., “Cloning Strategies inAspergillusfor Enzume Production: Prochymosin as a Model Sustem,” Proc. of Bio. Expo., 86:145-149 (1986).
Stohl, et al., “Construction of a Shuttle Vector for the Filamentous FungusNeurospora Crassa,” Proc. Natl. Acad. Sci. USA, 80:1058-1062 (1983).
Ballance, et al., “Transformation ofAspergillus Nidulansby the Orotidine-′5-Phosphate Decarboxylase Gene,” Biochemical and Biophysical Research Communications, 112(1):284-289 (1983).
Tilburn, et al.
Berka Randy Michael
Cullen Daniel
Gray Gregory Lawrence
Hayenga Kirk James
Lawlis Virgil Bryan
Flehr Hohbach Test Albritton & Herbert LLP
Genencor International Inc.
Schwartzman Robert A.
Shibuya Mark L.
Trecartin Richard F.
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