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
2001-03-06
2003-09-02
McKelvey, Terry (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...
C435S245000, C435S320100, C536S024100
Reexamination Certificate
active
06613547
ABSTRACT:
BACKGROUND OF THE INVENTION
Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand,
Mycotaxon
35:201-204, 1989; Kurtzman,
Mycologia
15 84:72-76, 1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.
Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems for several reasons. First, some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Second, recombinant expression cassettes are genomically integrated and therefore mitotically stable. Third, these yeasts are capable of secreting large amounts of recombinant proteins. See, for example, Faber et al.,
Yeast
11:1331, 1995; Romanos et al.,
Yeast
8:423, 1992; Cregg et al.,
Bio/Technology
11:905, 1993; U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; and U.S. Pat. No. 4,929,555; and Raymond, U.S. Pat. Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.
Previously described expression systems for methylotrophic yeasts rely largely on the use of methanol-inducible transcription promoters. The use of methanol-induced promoters is, however, problematic as production is scaled up to commercial levels. The overall volume of methanol used during the fermentation process can be as much as 40% of the final fermentation volume, and at 1000-liter fermentation scale and above the volumes of methanol required for induction necessitate complex and potentially expensive considerations.
There remains a need in the art for additional materials and methods to enable the use of methylotrophic yeasts for production of polypeptides of economic importance, including industrial enzymes and pharmaceutical proteins. The present invention provides such materials and methods as well as other, related advantages.
SUMMARY OF THE INVENTION
Within one aspect, the present invention provides an isolated DNA molecule of up to 1500 nucleotides in length comprising nucleotide 810 to nucleotide 1724 of SEQ ID NO:1.
Within a second aspect of the invention there is provided a DNA construct comprising the following operably linked elements: a first DNA segment comprising at least a portion of the sequence of SEQ ID NO: 1 from nucleotide 733 to nucleotide 1732, wherein the portion is a functional transcription promoter; a second DNA segment encoding a protein of interest other than a
Pichia methanolica
glyceraldehyde-3-phosphate dehydrogenase; and a third DNA segment comprising a transcription terminator. Within one embodiment, the first DNA segment is from 900 to 1500 nucleotides in length. Within another embodiment, the first DNA segment is from 900 to 1000 nucleotides in length. Within a further embodiment, the first DNA segment comprises nucleotide 810 to nucleotide 1724 of SEQ ID NO:1. Within an additional embodiment, the first DNA segment is essentially free of DNA encoding a
P. methanolica
glyceraldehyde-3-phosphate dehydrogenase. The DNA construct may further comprise a selectable marker, such as a
P. methanolica
gene, for example a
P. methanolica
ADE2 gene. The DNA construct may be a closed, circular molecule or a linear molecule. Within other embodiments, the DNA constuct further comprises a secretory signal sequence, such as a
Saccharomyces cerevisiae
alpha-factor pre-pro sequence, operably linked to the first and second DNA segments. Within additional embodiments, the third DNA segment comprises a transcription terminator of a
P. methanolica
AUG1 or GAP1 gene.
Within a third aspect of the invention there is provided a
P. methanolica
cell containing a DNA construct as disclosed above. Within one embodiment, the DNA construct is genomically integrated. Within a related embodiment, the DNA construct is genomically integrated in multiple copies. Within a further embodiment, the
P. methanolica
cell is functionally deficient in vacuolar proteases proteinase A and proteinase B.
Within a fourth aspect of the invention there is provided a method of producing a protein of interest comprising the steps of (a) culturing a
P. methanolica
cell as disclosed above whereby the second DNA segment is expressed and the protein of interest is produced, and (b) recovering the protein of interest.
Within a fifth aspect of the invention there is provided a DNA construct comprising the following operably linked elements: a first DNA segment comprising a
P. methanolica
gene transcription promoter; a second DNA segment encoding a protein of interest other than a
P. methanolica
protein; and a third DNA segment comprising nucleotides 2735 to 2795 of SEQ ID NO: 1.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and the attached drawings.
REFERENCES:
patent: 6258559 (2001-07-01), Zamost
patent: WO 97/17450 (1997-05-01), None
patent: WO 98/20035 (1998-05-01), None
Miller Brady G.
Raymond Christopher K.
Sloan James S.
Vanaja Erica
McKelvey Terry
Parker Gary E.
Walsh Brian J.
ZymoGenetics Inc.
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