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
2001-07-26
2003-11-11
Guzo, David (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...
C435S041000, C435S069900, C435S071100, C435S471000, C435S483000, C435S243000, C435S254100, C435S255100, C435S255300, C435S320100, C435S254110, C435S254200, C536S023100, C536S024100
Reexamination Certificate
active
06645739
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to expression systems for the production of polypeptides in yeast, to components thereof, and methods of making and using the same.
BACKGROUND OF THE INVENTION
The development of recombinant DNA technology has made possible the production of an enormous variety of useful polypeptides using microorganisms. For example, eukaryotic polypeptides such as human growth hormone, leukocyte interferons, human insulin and human proinsulin have been produced by various microorganisms including bacteria and a variety of yeasts. It is expected that the future will bring the production of polypeptides from a variety of other microorganisms through such recombinant DNA techniques.
Traditionally, commercial efforts employing recombinant DNA technology for the production of polypeptides have focused on the use of
Escherichia coli
(
E. coli
) as a host organism. However,
E. coli
has proved to be an unsuitable host in many situations. For example,
E. coli
contains a number of toxic pyrogenic factors that must be eliminated from any polypeptide to be used as a pharmaceutical product. The efficiency with which this purification can be achieved varies, of course, with the particular polypeptide. In addition, the proteolytic activities of
E. coli
can seriously limit yields of some useful products. These and other considerations have led to increased interest in alternative hosts, in particular, the use of eukaryotic organisms for the production of polypeptide products.
The production of polypeptide products in eukaryotic systems, e.g., yeast, provides for significant advantages relative to the use of prokaryotic systems such as
E. coli
for the production of polypeptides. For example, yeast have been employed in large scale fermentations for centuries, as compared to the relatively recent advent of large scale
E. coli
fermentations. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing. U.S. Pat. No. 4,414,329 discusses the growth of yeast such as
Pichia pastoris
(
P. pastoris
) to ultra-high cell densities, i.e., cell densities in excess of 100 g/L. U.S. Pat. No. 5,002,876 discusses the production of human tumor necrosis factor is
P. pastoris
. Yeast hosts are also advantageous in that many critical functions of the organism, e.g., oxidative phosphorylation, are performed within organelles, and hence not exposed to the possible deleterious effects of the organism's production of polypeptides foreign to the wild-type host cells. In addition, as an eukaryotic organism, yeast may prove capable of glycosylating expressed polypeptide products where such glycosylation is important to the bioactivity of the polypeptide product. It is also possible that as a eukaryotic organism, yeast will exhibit the same codon preferences as higher organisms, thus tending toward more efficient production of expression products from mammalian genes or from complementary DNA obtained by reverse transcription from, for example, mammalian messenger RNA (“mRNA”).
Methanol assimilating yeasts have been identified as attractive candidates for use in recombinant expression systems. Methanol assimilating yeasts are able to utilize methanol as a source of carbon and energy and can provide several advantages when used in expression systems. For example, some methanol assimilating yeasts grow rapidly on minimal defined media. In addition, certain genes of these yeast are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that promoters of these genes might be useful for the production of polypeptides of commercial value. Faber et al., Yeast 11:1331 (1995); Cregg et al, Bio/Technology 11:905 (1993), the disclosures of which are hereby incorporated by reference herein in their entirety.
Yeasts having the biochemical pathways necessary for methanol assimilation have traditionally been classified in four genera: Hansenula, Pichia, Candida and Torulopsis. Expression systems have been described using
P. pastoris
and
Hansenula polymorpha
. Faber et al., Curr. Genet. 25:305-10 (1994); Cregg et al., supra; Romanos et al., Yeast 2:423 (1992), the disclosures of which are hereby incorporated by reference herein in their entirety. However, these genera are based on cell morphology and growth characteristics, and do not reflect close genetic relationships. Moreover, it has been shown that not all species within these genera are capable of utilizing methanol as a source of carbon and energy. In addition, an examination of the phylogenetic relationship of several methanol-assimilating yeasts by partial sequencing of 18S and 26S ribosomal RNAs has shown that
Pichia pastoris
has significant base differences from other species of Pichia. See Yamada et al., Biosci. Biotech. Biochem. 59(3):439-44 (1995), the disclosure of which is hereby incorporated by reference herein in its entirety. There may thus be substantial differences in physiology and metabolism between individual species of a genus, indicating that even members of the same genus may have differing characteristics not easily predictable based on phylogenetic characteristics.
The development of these poorly characterized yeast species for use in expression systems has been severely hampered by the lack of knowledge about transformation techniques and conditions, especially the particular regulatory regions for each strain. Depending on the strain of yeast used and the specific transformation technique, from about 50 to about 100,000 transformants per microgram of plasmid are obtained (See, Dohmen et al., Yeast 7, 691-2 (1991). Although
Saccharomyces cervisiae
and
Candida boidinii
can be transformed using lithium acetate, this method does not work well in
Pichia pastoris
. Furthermore, even using the same strain of yeast and the same transformation method, different plasmids yield different efficiencies. Development has also been slowed in part by a lack of suitable materials such as vectors, promoters, selectable markers and host cells. In addition, it has been shown that auxotrophic mutations are often not available, precluding direct selection for transformants by auxotrophic complementation. For recombinant DNA technology to fully sustain its promise, new host/vector systems must be devised which facilitate the manipulation of DNA as well as optimize expression of inserted DNA sequences so that the desired polypeptide products can be prepared under controlled conditions and in high yield.
There is a need for novel expression systems that include certain strains of yeast as hosts for the production of polypeptide products. The present invention is directed to these, as well as other, important ends.
SUMMARY OF THE INVENTION
The present invention relates to yeast-based expression systems for the production of desired polypeptides. The expression systems comprise a yeast host cell, selected from the group consisting of
Ogataea wickerhamii
(
O. wickerhamii
),
Ogataea kodamae
(
O. kodamae
),
Ogataea pini
(
O. pini
),
Komagataella pastoris
(
K. pastoris
), or
Zygosaccharomyces pastori
(
Z. pastori
) and a vector. The vector is adapted to express a nucleic acid molecule encoding a desired polypeptide, which nucleic acid molecule is operably linked to one or more regulatory regions. In some preferred embodiments the yeast host cell is
K. pastoris.
The present invention also relates to methods for isolating a desired polypeptide from a yeast host cell. The methods comprise transforming a yeast cell with an expression vector comprising a nucleic acid molecule encoding a desired polypeptide, transforming the host cell with said expression vector, expanding the transformed host cells in culture, and isolating the desired polypeptide from the culture. In some preferred embodiments, the yeast host cell is
K. pastoris
. In some preferred embodiments, the vector comprises a
K. pastoris
alcohol oxidase gene promoter.
The present invention further relates to an isolated nucleic acid molecule hav
Guzo David
Lambertson David A.
Phoenix Pharmacologies, Inc.
Woodcock & Washburn LLP
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