U.S. patent application number 10/013784 was filed with the patent office on 2002-07-04 for pichia methanolica glyceraldehyde-3-phosphate dehydrogenase 2 promoter and terminator.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Raymond, Christopher K..
Application Number | 20020086366 10/013784 |
Document ID | / |
Family ID | 26849818 |
Filed Date | 2002-07-04 |
United States Patent
Application |
20020086366 |
Kind Code |
A1 |
Raymond, Christopher K. |
July 4, 2002 |
PICHIA METHANOLICA GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE 2
PROMOTER AND TERMINATOR
Abstract
Transcription promoter and terminator sequences from the Pichia
methanolica glyceraldehyde-3-phosphate dehydrogenase 2 gene (GAP2
gene) are disclosed. The sequences are useful within DNA constructs
for the production of proteins of interest in cultured P.
methanolica cells. Within the expression vectors, a GAP2 promoter
and/or a GAP2 terminator is operably linked to a DNA segment
encoding the protein of interest.
Inventors: |
Raymond, Christopher K.;
(Seattle, WA) |
Correspondence
Address: |
Gary E. Parker
Patent Department, ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26849818 |
Appl. No.: |
10/013784 |
Filed: |
December 7, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10013784 |
Dec 7, 2001 |
|
|
|
09653403 |
Sep 1, 2000 |
|
|
|
60152744 |
Sep 8, 1999 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/254.23; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12N 15/81 20130101;
C07K 14/39 20130101 |
Class at
Publication: |
435/69.1 ;
435/254.23; 435/320.1; 536/23.2 |
International
Class: |
C12P 021/02; C07H
021/04; C12N 001/18 |
Claims
What is claimed is:
1. An isolated DNA molecule of up to 5000 nucleotides in length
comprising nucleotide 93 to nucleotide 1080 of SEQ ID No:1.
2. 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 93 to nucleotide 1092,
wherein said 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.
3. The DNA construct of claim 2 wherein said first DNA segment is
from 900 to 1500 nucleotides in length.
4. The DNA construct of claim 2 wherein the first DNA segment
comprises nucleotide 93 to nucleotide 1080 of SEQ ID No:1.
5. The DNA construct of claim 2 wherein the first DNA segment is
substantially free of Pichia methanolica glyceraldehyde-3-phosphate
dehydrogenase gene coding sequence.
6. The DNA construct of claim 2, further comprising a selectable
marker.
7. The DNA construct of claim 2, further comprising a secretory
signal sequence operably linked to the first and second DNA
segments.
8. The DNA construct of claim 7, wherein the secretory signal
sequence is the S. Cerevisiae alpha-factor pre-pro sequence.
9. The DNA construct of claim 2 wherein said third DNA segment
comprises a transcription terminator of a Pichia methanolica AUG1
or GAP2 gene.
10. The DNA construct of claim 9, wherein said terminator comprises
nucleotides 2095 to 2145 of SEQ ID NO:1.
11. A Pichia methanolica cell containing the DNA construct of claim
2.
12. The Pichia methanolica cell of claim 11 wherein the DNA
construct is genomically integrated.
13. The Pichia methanolica cell of claim 12 wherein the DNA
construct is genomically integrated in multiple copies.
14. The Pichia methanolica cell of claim 11 wherein the first DNA
segment is from 900 to 1500 nucleotides in length.
15. The Pichia methanolica cell of claim 11 wherein the first DNA
segment comprises nucleotide 93 to nucleotide 1080 of SEQ ID
NO:1.
16. The Pichia methanolica cell of claim 11, wherein the cell is
functionally deficient in vacuolar proteases proteinase A and
proteinase B.
17. A method of producing a protein of interest comprising:
culturing the cell of claim 11 whereby the second DNA segment is
expressed and the protein of interest is produced; and recovering
the protein of interest from the cultured cell.
18. The method of claim 17 wherein the DNA construct is genomically
integrated in multiple copies.
19. The method of claim 17, wherein the cell is deficient in
vacuolar proteases proteinase A and proteinase B.
20. A DNA construct comprising the following operably linked
elements: a first DNA segment comprising a Pichia methanolica gene
transcription promoter; a second DNA segment encoding a protein of
interest other than a Pichia methanolica protein; and a third DNA
segment comprising nucleotides 2095 to 2145 of SEQ ID NO:2.
Description
BACKGROUND OF THE INVENTION
[0001] 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 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.
[0002] 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.
[0003] 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 volue, and at
1000-liter fermentation scale and above the volumes of methanol
required for induction necessitate complex and potentially
expensive considerations.
[0004] 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
[0005] It is an object of the present invention to provide
transcription promoter and terminator sequences for use in Pichia
methanolica. It is a further object of the invention to provide
materials and methods for obtaining constitutive expression of
heterologous DNA in P. methanolica. It is also an object of the
invention to provide methods for production of polypeptides in P.
methanolica, which methods can be readily scaled up to industrial
levels, and to provide materials that can be used within these
methods. It is another object of the invention to provide materials
and methods for obtaining constitutive transcription of
heterologous DNA to produce recombinant proteins in P.
methanolica.
[0006] Within one aspect, the present invention provides an
isolated DNA molecule of up to 5000 nucleotides in length
comprising nucleotide 93 to nucleotide 1080 of SEQ ID NO:1.
[0007] 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 93 to nucleotide 1092, 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 an additional embodiment,
the first DNA segment is substantially free of Pichia methanolica
glyceraldehyde-3-phosphate dehydrogenase gene coding sequence. The
DNA construct may further comprise a selectable marker, preferably
a Pichia methanolica gene, more preferably a Pichia methanolica
ADE2 gene. The DNA construct may be a closed, circular molecule or
a linear molecule. Within other embodiments, the DNA construct
further comprises a secretory signal sequence, such as the S.
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 Pichia
methanolica AUG1 or GAP2 gene.
[0008] Within a third aspect of the invention there is provided a
Pichia 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.
[0009] 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 from the
cultured cell.
[0010] 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 Pichia methanolica gene
transcription promoter; a second DNA segment encoding a protein of
interest other than a Pichia methanolica protein; and a third DNA
segment comprising nucleotides 2095 to 2145 of SEQ ID NO:1.
[0011] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0012] The term "allelic variant" is used herein to denote an
alternative form of a gene. Allelic variation is known to exist in
populations and arises through mutation.
[0013] A "DNA construct" is a DNA molecule, either single- or
double-stranded, that has been modified through human intervention
to contain segments of DNA combined and juxtaposed in an
arrangement not existing in nature.
[0014] A "DNA segment" is a portion of a larger DNA molecule having
specified attributes. For example, a DNA segment encoding a
specified polypeptide is a portion of a longer DNA molecule, such
as a plasmid or plasmid fragment, that, when read from the 5' to
the 3' direction, encodes the sequence of amino acids of the
specified polypeptide.
[0015] The term "functionally deficient" denotes the expression in
a cell of less than 10% of an activity as compared to the level of
that activity in a wild-type counterpart. It is preferred that the
expression level be less than 1% of the activity in the wild-type
counterpart, more preferably less than 0.01% as determined by
appropriate assays. It is most preferred that the activity be
essentially undetectable (i.e., not significantly above
background). Functional deficiencies in genes can be generated by
mutations in either coding or non-coding regions.
[0016] The term "gene" is used herein to denote a DNA segment
encoding a polypeptide. Where the context allows, the term includes
genomic DNA (with or without intervening sequences), cDNA, and
synthetic DNA. Genes may include non-coding sequences, including
promoter elements.
[0017] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include CDNA and genomic clones.
[0018] "Operably linked", when referring to DNA segments, indicates
that the segments are arranged so that they function in concert for
their intended purposes, e.g., transcription initiates in the
promoter and proceeds through the coding segment to the
terminator.
[0019] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When these terms are applied to
double-stranded molecules they are used to denote overall length
and will be understood to be equivalent to the term "base pairs".
It will be recognized by those skilled in the art that the two
strands of a double-stranded polynucleotide may differ slightly in
length and that the ends thereof may be staggered as a result of
enzymatic cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0020] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0021] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes. Sequences within
promoters that function in the initiation of transcription are
often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, and transcription factor binding sites. See, in general,
Watson et al., eds., Molecular Biology of the Gene, 4th ed., The
Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.,
1987.
[0022] A "pro sequence" is a DNA sequence that commonly occurs
immediately 5' to the mature coding sequence of a gene encoding a
secretory protein.
[0023] The pro sequence encodes a pro peptide that serves as a
cis-acting chaperone as the protein moves through the secretory
pathway.
[0024] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are commonly defined in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0025] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway. A secretory
peptide and a pro peptide may be collectively referred to as a
pre-pro peptide.
[0026] All references cited herein are incorporated by reference in
their entirety.
[0027] The present invention provides isolated DNA molecules
comprising a Pichia methanolica glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene promoter. The invention also provides
isolated DNA molecules comprising a P. methanolica GAPDH gene
terminator. The promoter and terminator can be used within methods
of producing proteins of interest, including proteins of
pharmaceutical or industrial value.
[0028] The sequence of a DNA molecule comprising a Pichia
methanolica glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene
promoter, coding region, and terminator is shown in SEQ ID NO:1.
The gene has been designated GAP2. Those skilled in the art will
recognize that SEQ ID NO:1 represents a single allele of the P.
methanolica GAP2 gene and that other functional alleles (allelic
variants) are likely to exist, and that allelic variation may
include nucleotide changes in the promoter region, coding region,
or terminator region.
[0029] The partial sequence of a second P. methanolica
glyceraldehyde-3-phosphate dehydrogenase gene, designated GAP1, is
shown in SEQ ID NO:2.
[0030] Within SEQ ID NO:1, the GAPDH open reading frame begins with
the methionine codon (ATG) at nucleotides 1093-1095. The
transcription promoter is located upstream of the ATG. Gene
expression experiments showed that a functional promoter was
contained within the ca. 1000 nucleotide 5'-flanking region of the
GAP2 gene.
[0031] Preferred portions of the sequence shown in SEQ ID NO:1 for
use within the present invention as transcription promoters include
segments comprising at least 900 contiguous nucleotides of the 5'
non-coding region of SEQ ID NO:1, and preferably comprising
nucleotide 93 to nucleotide 1080 of the sequence shown in SEQ ID
NO:1. Those skilled in the art will recognize that longer portions
of the 5' non-coding region of the P. methanolica GAP2 gene can
also be used. Promoter sequences of the present invention can thus
include the sequence of SEQ ID NO:1 through nucleotide 1092 in the
3' direction and can extend to or beyond nucleotide 1 in the 5'
direction. In general, the promoter used within an expression DNA
construct will not exceed 1.5 kb in length, and will preferably not
exceed 1.0 kb in length. In addition to these promoter fragments,
the invention also provides isolated DNA molecules of up to about
3300 bp, as well as isolated DNA molecules of up to 5000 bp,
wherein said molecules comprise the P. methanolica GAP2 promoter
sequence.
[0032] As disclosed in more detail in the examples that follow, the
sequence of SEQ ID NO:1 from nucleotide 93 to nucleotide 1080
provides a functional transcription promoter. However, additional
nucleotides can be removed from either or both ends of this
sequence and the resulting sequence tested for promoter function by
joining it to a sequence encoding a protein, preferably a protein
for which a convenient assay is readily available.
[0033] Within the present invention it is preferred that the GAP2
promoter be substantially free of GAP2 gene coding sequence, which
begins with nucleotide 1093 in SEQ ID NO:1. As used herein,
"substantially free" of GAP2 gene coding sequence means that the
promoter DNA includes not more than 15 nucleotides of the GAP2
coding sequence, preferably not more than 10 nucleotides, and more
preferably not more than 3 nucleotides. Within a preferred
embodiment of the invention, the GAP2 promoter is provided free of
coding sequence of the P. methanolica GAP2 gene. However, those
skilled in the art will recognize that a GAP2 gene fragment that
includes the initiation ATG (nucleotides 1093 to 1095) of SEQ ID
NO:1 can be operably linked to a heterologous coding sequence that
lacks an ATG, with the GAP2 ATG providing for intition of
translation of the heterologous sequence. Those skilled in the art
will further recognize that additional GAP2 coding sequences can
also be included, whereby a fusion protein comprising GAP2 and
heterologous amino acid sequences is produced. Such a fusion
protein may comprise a cleavage site to facilitate separation of
the GAP2 and heterologous sequences subsequent to translation.
[0034] In addition to the GAP2 promoter sequence, the present
invention also provides transcription terminator sequences derived
from the 3' non-coding region of the P. methanolica GAP2 gene. A
consensus transcription termination sequence (Chen and Moore, Mol.
Cell. Biol. 12:3470-3481, 1992) is at nucleotides 2136 to 2145 of
SEQ ID NO:1. Within the present invention, there are thus provided
transcription terminator gene segments of at least about 50 bp,
preferably at least 60 bp, more preferably at least 90 bp, still
more preferably about 200 bp in length. The terminator segments of
the present invention may comprise 500-1000 nucleotides of the 3'
non-coding region of SEQ ID NO:1. These segments comprise the
termination sequence disclosed above, and preferably have as their
5' termini nucleotide 2095 of SEQ ID NO:1. Those skilled in the art
will recognize, however, that the transcription terminator segment
that is provided in an expression vector can include at its 5'
terminus the TAA translation termination codon at nucleotides
2092-2094 of SEQ ID No:1 to permit the insertion of coding
sequences that lack a termination codon.
[0035] Techniques for manipulating cloned DNA molecules and
introducing exogenous DNA into a variety of host cells are well
known in the art and are disclosed by, for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Murray,
ed., Gene Transfer and Expression Protocols, Humana Press, Clifton,
N.J., 1991; Glick and Pasternak, Molecular Biotechnology:
Principles and Applications of Recombinant DNA, ASM Press,
Washington, D.C., 1994; Ausubel et al. (eds.), Short Protocols in
Molecular Biology, 3rd edition, John Wiley and Sons, Inc., N.Y.,
1995; Wu et al., Methods in Gene Biotechnology, CRC Press, New
York, 1997. DNA vectors, including expression vectors, commonly
contain a selectable marker and origin of replication that function
in a bacterial host (e.g., E. coli) to permit the replication and
amplification of the vector in a prokaryotic host. If desired,
these prokaryotic elements can be removed from a vector before it
is introduced into an alternative host. For example, such
prokaryotic sequences can be removed by linearization of the vector
prior to its introduction into a P. methanolica host cell.
[0036] Within certain embodiments of the invention, expression
vectors are provided that comprise a first DNA segment comprising
at least a portion of the sequence of SEQ ID No:1 that is a
functional transcription promoter operably linked to a second DNA
segment encoding a protein of interest. When it is desired to
secrete the protein of interest, the vector will further comprise a
secretory signal sequence operably linked to the first and second
DNA segments. The secretory signal sequence may be that of the
protein of interest, or may be derived from another secreted
protein, preferably a secreted yeast protein. A preferred such
yeast secretory signal sequence is the S. Cerevisiae alpha-factor
(MF.alpha.1) pre-pro sequence (disclosed by Kurjan et al., U.S.
Pat. No. 4,546,082 and Brake, U.S. Pat. No. 4,870,008).
[0037] Within other embodiments of the invention, expression
vectors are provided that comprise a DNA segment comprising a
portion of SEQ ID No:1 that is a functional transcription
terminator operably linked to an additional DNA segment encoding a
protein of interest. Within one embodiment, the GAP2 promoter and
terminator sequences of the present invention are used in
combination, wherein both are operably linked to a DNA segment
encoding a protein of interest within an expression vector.
[0038] Expression vectors of the present invention further comprise
a selectable marker to permit identification and selection of P.
methanolica cells containing the vector. Selectable markers provide
for a growth advantage of cells containing them. The general
principles of selection are well known in the art. The selectable
marker is preferably a P. methanolica gene. Commonly used
selectable markers are genes that encode enzymes required for the
synthesis of amino acids or nucleotides. Cells having mutations in
these genes cannot grow in media lacking the specific amino acid or
nucleotide unless the mutation is complemented by the selectable
marker. Use of such "selective" culture media ensures the stable
maintenance of the heterologous DNA within the host cell. A
preferred selectable marker of this type for use in P. methanolica
is a P. methanolica ADE2 gene, which encodes
phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21).
See, Raymond, U.S. Pat. No. 5,736,383. The ADE2 gene, when
transformed into an ade2 host cell, allows the cell to grow in the
absence of adenine. The coding strand of a representative P.
methanolica ADE2 gene sequence is shown in SEQ ID NO:3. The
sequence illustrated includes 1006 nucleotides of 5' non-coding
sequence and 442 nucleotides of 3' non-coding sequence, with the
initiation ATG codon at nucleotides 1007-1009. Within a preferred
embodiment of the invention, a DNA segment comprising nucleotides
407-2851 is used as a selectable marker, although longer or shorter
segments could be used as long as the coding portion is operably
linked to promoter and terminator sequences. In the alternative, a
dominant selectable marker, which provides a growth advantage to
wild-type cells, may be used. Typical dominant selectable markers
are genes that provide resistance to antibiotics, such as
neomycin-type antibiotics (e.g., G418), hygromycin B, and
bleomycin/phleomycin-type antibiotics (e.g., Zeocin.TM.; available
from Invitrogen Corporation, San Diego, Calif.). A preferred
dominant selectable marker for use in P. methanolica is the Sh bla
gene, which inhibits the activity of Zeocin.TM..
[0039] The use of P. methanolica cells as a host for the production
of recombinant proteins is disclosed in WIPO Publications WO
97/17450, WO 97/17451, WO 98/02536, and WO 98/02565; and U.S. Pat.
Nos. 5,716,808, 5,736,383, 5,854,039, and 5,736,383. Expression
vectors for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. To facilitate
integration of the expression vector DNA into the host chromosome,
it is preferred to have the entire expression segment of the
plasmid flanked at both ends by host DNA sequences (e.g., AUG1 3'
sequences). Electroporation is used to facilitate the introduction
of a plasmid containing DNA encoding a polypeptide of interest into
P. methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (.tau.) of from 1
to 40 milliseconds, most preferably about 20 milliseconds.
[0040] Integrative transformants are preferred for use in protein
production processes. Such cells can be propagated without
continuous selective pressure because DNA is rarely lost from the
genome. Integration of DNA into the host chromosome can be
confirmed by Southern blot analysis. Briefly, transformed and
untransformed host DNA is digested with restriction endonucleases,
separated by electrophoresis, blotted to a support membrane, and
probed with appropriate host DNA segments. Differences in the
patterns of fragments seen in untransformed and transformed cells
are indicative of integrative transformation. Restriction enzymes
and probes can be selected to identify transforming DNA segments
(e.g., promoter, terminator, heterologous DNA, and selectable
marker sequences) from among the genomic fragments.
[0041] Differences in expression levels of heterologous proteins
can result from such factors as the site of integration and copy
number of the expression cassette among individual isolates. It is
therefore advantageous to screen a number of isolates for
expression level prior to selecting a production strain. Isolates
exhibiting a high expression level will commonly contain multiple
integrated copies of the desired expression cassette. A variety of
suitable screening methods are available. For example, transformant
colonies are grown on plates that are overlayed with membranes
(e.g., nitrocellulose) that bind protein. Proteins are released
from the cells by secretion or following lysis, and bind to the
membrane. Bound protein can then be assayed using known methods,
including immunoassays. More accurate analysis of expression levels
can be obtained by culturing cells in liquid media and analyzing
conditioned media or cell lysates, as appropriate. Methods for
concentrating and purifying proteins from media and lysates will be
determined in part by the protein of interest. Such methods are
readily selected and practiced by the skilled practitioner.
[0042] For production of secreted proteins, host cells having
functional deficiencies in the vacuolar proteases proteinase A,
which is encoded by the PEP4 gene, and proteinase B, which is
encoded by the PRB1 gene, are preferred in order to minimize
spurious proteolysis. Vacuolar protease activity (and therefore
vacuolar protease deficiency) is measured using any of several
known assays. Preferred assays are those developed for
Saccharomyces cerevisiae and disclosed by Jones, Methods Enzymol.
194:428-453, 1991. A preferred such assay is the APNE overlay
assay, which detects activity of carboxypeptidase Y (CpY). See,
Wolf and Fink, J. Bact. 123:1150-1156, 1975. Because the zymogen
(pro)CpY is activated by proteinase A and proteinase B, the APNE
assay is indicative of vacuolar protease activity in general. The
APNE overlay assay detects the carboxypeptidase Y-mediated release
of .beta.-naphthol from
N-acetyl-phenylalanine-.beta.-naphthyl-ester (APNE), which results
in the formation of an isoluble red dye by the reaction of the
P-naphthol with the diazonium salt Fast Garnet GBC. Cells growing
on assay plates (YEPD plates are preferred) at room temperature are
overlayed with 8 ml RxM. RxM is prepared by combining 0.175 g agar,
17.5 ml H.sub.2O, and 5 ml 1 M Tris-HCl pH 7.4, microwaving the
mixture to dissolve the agar, cooling to .about.55.degree. C.,
adding 2.5 ml freshly made APNE (2 mg/ml in dimethylformamide)
(Sigma Chemical Co., St. Louis, Mo.), and, immediately before
assay, 20 mg Fast Garnet GBC salt (Sigma Chemical Co.). The overlay
is allowed to solidify, and color development is observed.
Wild-type colonies are red, whereas CPY deletion strains are white.
Carboxypeptidase Y activity can also be detected by the well test,
in which cells are distributed into wells of a microtiter test
plate and incubated in the presence of N-benzoyl-L-tyrosine
p-nitroanilide (BTPNA) and dimethylformamide. The cells are
permeabilized by the dimethylformamide, and CpY in the cells
cleaves the amide bond in the BTPNA to give the yellow product
p-nitroaniline. Assays for CpY will detect any mutation that
reduces protease activity so long as that activity ultimately
results in the reduction of CpY activity.
[0043] P. methanolica cells are cultured in a medium comprising
adequate sources of carbon, nitrogen and trace nutrients at a
temperature of about 25.degree. C. to 35.degree. C. Liquid cultures
are provided with sufficient aeration by conventional means, such
as shaking of small flasks or sparging of fermentors. A preferred
culture medium for P. methanolica is YEPD (2% D-glucose, 2%
Bacto.TM. Peptone (Difco Laboratories, Detroit, Mich.), 1%
Bacto.TM. yeast extract (Difco Laboratories), 0.004% adenine,
0.006% L-leucine).
[0044] For large-scale culture, one to two colonies of a P.
methanolica strain can be picked from a fresh agar plate (e.g, YEPD
agar) and suspended in 250 ml of YEPD broth contained in a
two-liter baffled shake flask. The culture is grown for 16 to 24
hours at 30.degree. C. and 250 rpm shaking speed. Approximately 50
to 80 milliliters of inoculum are used per liter starting fermentor
volume (5-8% v/v inoculum).
[0045] A preferred fermentation medium is a soluble medium
comprising glucose as a carbon source, inorganic ammonia,
potassium, phosphate, iron, and citric acid. As used herein, a
"soluble medium" is a medium that does not contain visible
precipitation. Preferably, the medium lacks phosphate glass (sodium
hexametaphosphate). A preferred medium is prepared in deionized
water and does not contain calcium sulfate. As a minimal medium, it
is preferred that the medium lacks polypeptides or peptides, such
as yeast extracts. However, acid hydrolyzed casein (e.g., casamino
acids or amicase) can be added to the medium if desired. An
illustrative fermentation medium is prepared by mixing the
following compounds: (NH.sub.4).sub.2SO.sub.4 (11.5 grams/liter),
K.sub.2HPO.sub.4 (2.60 grams/liter), KH.sub.2PO.sub.4 (9.50
grams/liter), FeSO.sub.4. 7H.sub.2O (0.40 grams/liter), and citric
acid (1.00 gram/liter). After adding distilled, deionized water to
one liter, the solution is sterilized by autoclaving, allowed to
cool, and then supplemented with the following: 60% (w/v) glucose
solution (47.5 milliliters/liter), 10.times. trace metals solution
(20.0 milliliters/liter), 1 M MgSO.sub.4 (20.0 milliliters/liter),
and vitamin stock solution (2.00 milliliters/liter). The 10.times.
trace metals solution contains FeSO.sub.4. 7H.sub.2O (100 mM),
CuSO.sub.4. 5H.sub.2O (2 mM), ZnSO.sub.4. 7H.sub.2O (8 mM),
MnSO.sub.4. H.sub.2O (8 mM), CoCl.sub.2e6H.sub.20 (2 mM),
Na.sub.2MoO.sub.4e2H.sub.20 (1 mM), H.sub.3BO.sub.3 (8 mM), KI (0.5
mM), NiSO.sub.4. 6H.sub.2O (1 mM), thiamine (0.50 grams/liter), and
biotin (5.00 milligrams/liter). The vitamin stock solution contains
inositol (47.00 grams/liter), pantothenic acid (23.00 grams/liter),
pyrodoxine (1.20 grams/liter), thiamine (5.00 grams/liter), and
biotin (0.10 gram/liter). Those of skill in the art can vary these
particular ingredients and amounts. For example, ammonium sulfate
can be substituted with ammonium chloride, or the amount of
ammonium sulfate can be varied, for example, from about 11 to about
22 grams/liter.
[0046] After addition of trace metals and vitamins, the pH of the
medium is typically adjusted to pH 4.5 by addition of 10%
H.sub.3PO.sub.4. Generally, about 10 milliliters/liter are added,
and no additional acid addition will be required. During
fermentation, the pH is maintained between about 3.5 to about 5.5,
or about 4.0 to about 5.0, depending on protein produced, by
addition of 5 N NH.sub.4OH.
[0047] An illustrative fermentor is a BIOFLO 3000 fermentor system
(New Brunswick Scientific Company, Inc.; Edison, N.J.). This
fermentor system can handle either a six-liter or a fourteen-liter
fermentor vessel. Fermentations performed with the six-liter vessel
are prepared with three liters of medium, whereas fermentations
performed with the fourteen-liter vessel are prepared with six
liters of medium. The fermentor vessel operating temperature is
typically set to 30.degree. C. for the course of the fermentation,
although the temperature can range between 27-31.degree. C.
depending on the protein expressed. The fermentation is initiated
in a batch mode. The glucose initially present is often used by
approximately 10 hours elapsed fermentation time (EFT), at which
time a glucose feed can be initiated to increase the cell mass. An
illustrative glucose feed contains 900 milliliters of 60% (w/v)
glucose, 60 milliliters of 50% (w/v) (NH.sub.4)SO.sub.4, 60
milliliters of 10.times. trace metals solution, and 30 milliliters
of 1 M MgSO.sub.4. Pichia methanolica fermentation is robust and
requires high agitation, aeration, and oxygen sparging to maintain
the percentage dissolved oxygen saturation above 30%. The
percentage dissolved oxygen should not drop below 15% for optimal
expression and growth. The biomass typically reaches about 30 to
about 80 grams dry cell weight per liter at 48 hours EFT.
[0048] Proteins produced according to the present invention are
recovered from the host cells using conventional methods. If the
protein is produced intracellulary, the cells are harvested (e.g.,
by centrifugation) and lysed to release the cytoplasmic contents.
Methods of lysis include enzymatic and mechanical disruption. The
crude extract is then fractionated according to known methods, the
specifics of which will be determined for the particular protein of
interest. Secreted proteins are recovered from the conditioned
culture medium using standard methods, also selected for the
particular protein. See, in general, Scopes, Protein Purification:
Principles and Practice, Springer-Verlag, New York, 1994.
[0049] The materials and methods of the present invention can be
used to produce proteins of research, industrial, or pharmaceutical
interest. Such proteins include enzymes, such as lipases,
cellulases, and proteases; enzyme inhibitors, including protease
inhibitors; growth factors such as platelet derived growth factor
(PDGF), fibroblast growth factors (FGF), epidermal growth factor
(EGF), vascular endothelial growth factors (VEGFs); glutamic acid
decarboxylase (GAD); cytokines, such as erythropoietin,
thrombopoietin, colony stimulating factors, interleukins, and
interleukin antagonist; hormones, such as insulin, proinsulin,
leptin, and glucagon; and receptors, including growth factor
receptors, which can be expressed in truncated form ("soluble
receptors") or as fusion proteins with, for example, immunoglobulin
constant region sequences. DNAs encoding these and other proteins
are known in the art. See, for example, U.S. Pat. Nos. 4,889,919;
5,219,759; 4,868,119; 4,968,607; 4,599,311; 4,784,950; 5,792,850;
5,827,734; 4,703,008; 4,431,740; and 4,762,791; and WIPO
Publications WO 95/21920 and WO 96/22308.
[0050] It is particularly preferred to use the present invention to
produce unglycosylated pharmaceutical proteins. Yeast cells,
including P. methanolica cells, produce glycoproteins with
carbohydrate chains that differ from their mammalian counterparts.
Mammalian glycoproteins produced in yeast cells may therefore be
regarded as "foreign" when introduced into a mammal, and may
exhibit, for example, different pharmacokinetics than their
naturally glycosylated counterparts.
[0051] The invention is further illustrated by the following,
non-limiting examples.
EXAMPLES
Example 1
[0052] To clone the P. methanolica GAP1 gene, sense (ZC11,356; SEQ
ID NO:4) and antisense (ZC11,357; SEQ ID NO:5) PCR primers were
designed from an alignment of the coding regions of GAPDH genes of
Saccharomyces cerevisiae, Kluyveromyces lactis, and mouse. The
primers were then used to amplify P. methanolica genomic DNA. An
amplified sequence 608 bp long was recovered and was found to have
78.1% homology to the corresponding S. Cerevisiae GAPDH gene
sequence.
[0053] A P. methanolica genomic library was constructed in the
vector pRS426 (Christianson et al., Gene 110:119-122, 1992), a
shuttle vector comprising 2.mu. and S. cerevisiae URA3 sequences,
allowing it to be propagated in S. cerevisiae. Genomic DNA was
prepared from strain CBS6515 according to standard procedures.
Briefly, cells were cultured overnight in rich media, spheroplasted
with zymolyase, and lysed with SDS. DNA was precipitated from the
lysate with ethanol and extracted with a phenol/chloroform mixture,
then precipitated with ammonium acetate and ethanol. Gel
electrophoresis of the DNA preparation showed the presence of
intact, high molecular weight DNA and appreciable quantities of
RNA. The DNA was partially digested with Sau 3A by incubating the
DNA in the presence of a dilution series of the enzyme. Samples of
the digests were analyzed by electrophoresis to determine the size
distribution of fragments. DNA migrating between 4 and 12 kb was
cut from the gel and extracted from the gel slice. The
size-fractionated DNA was then ligated to pRS426 that had been
digested with Bam HI and treated with alkaline phosphatase.
Aliquots of the reaction mixture were electroporated into E. coli
MC 1061 cells using an electroporator (Gene Pulser.TM.; BioRad
Laboratories, Hercules, Calif.) as recommended by the
manufacturer.
[0054] The library was screened by PCR using sense (ZC11,733; SEQ
ID NO:6) and antisense (ZC11,734; SEQ ID NO:7) primers designed
from the sequenced region of the P. methanolica GAPDH. The PCR
reaction mixture was incubated for one minute at 94.degree. C.;
followed by 34 cycles of 94.degree. C., one minute, 52.degree. C.,
45 seconds, 72.degree. C., two minutes; and a termination cycle of
94.degree. C., one minute, 54.degree. C., one minute, 72.degree.
C., eleven minutes. Starting with 43 library pools, positive pools
were identified and broken down to individual colonies. A single
colony with a pRS426 plasmid containing the P. methanolica GAPDH
gene as its insert was isolated. The orientation of the GAPDH gene
and the length of the 5' and 3' flanking sequences in the insert
were deduced by DNA sequencing (SEQ ID NO:2). This gene was
designated GAP1.
[0055] Within SEQ ID NO:2, the GAPDH open reading frame begins with
the methionine codon (ATG) at nucleotides 1733-1735. The
transcription promoter is located upstream of the ATG. Gene
expression experiments showed that a functional promoter was
contained within the ca. 900 nucleotide 5'-flanking region of the
GAP1 gene. Analysis of this promoter sequence revealed the presence
of a number of sequences homologous to Saccharomyces cerevisiae
promoter elements. These sequences include a concensus TATAAA box
at nucleotides 1584 to 1591, a consensus Rap1p binding site (Graham
and Chambers, Nuc. Acids Res. 22:124-130, 1994) at nucleotides 1355
to 1367, and potential Gcr1p binding sites (Shore, Trends Genet.
10:408-412, 1994) at nucleotides 1225 to 1229, 1286 to 1290, 1295
to 1299, 1313 to 1317, 1351 to 1354, 1370 to 1374, 1389 to 1393,
and 1457 to 1461. A consensus transcription termination sequence
(Chen and Moore, Mol. Cell. Biol. 12:3470-3481, 1992) was
identified at nucleotides 2774 to 2787 of SEQ ID NO:2.
[0056] A plasmid containing the GAP1 gene, designated pGAPDH, has
been deposited as an E. coli strain MC1061 transformant with
American Type Culture Collection, Manassas, Va. under the terms of
the Budapest Treaty. The deposited strain has been assigned the
designation PTA-3 and a deposit date of May 4, 1999.
Example 2
[0057] Analysis of the P. methanolica genome by Southern blotting,
using a PCR product from the coding region of the cloned GAP1 gene
as a probe, indicated the presence of three independent GAPDH
genes. Primers designed from the cloned sequence were used in
various combinations to amplify P. methanolica genomic DNA.
Positive pools were screened by PCR, and positives were
re-amplified. PCR products were sequenced. Eight pools were found
to be the same and corresponded to the previously cloned GAP1 gene.
Two pools were distinct from the previously cloned gene and were
identical to each other. Each of these two pools was plated and
amplified by PCR through several rounds of sub-dividing. Sub-pools
were streaked, and single colonies were picked for a final round of
PCR screening. Positive clones were analyzed by PCR and restriction
digestion. Each clone was found to be carried on a .about.5 kb
genomic segment. This gene, which was designated GAP2, was
partially sequenced. The sequenced region included an open reading
frame of 1002 base pairs (including the termination codon), a 5'
non-coding region of 1092 base pairs, and a 3' non-coding region of
1239 base pairs (SEQ ID No:1).
Example 3
[0058] A fragment of GAP2 DNA (SEQ ID No:1) was isolated by PCR
using two primers. Primer ZC19,334 (SEQ ID NO:8) contained 26 bp of
vector flanking sequence and 25 bp corresponding to the 5' end of
the first 1000 bp of the GAP2 promoter. Primer ZC19,333 (SEQ ID
NO:9) contained 35 bp of the 3' end corresponding to S. Cerevisiae
alpha factor pre-pro sequence and 29 bp corresponding to the 3' end
of the GAP2 promoter. The latter primer altered the 5' flanking
sequence at nucleotides 1081-1092 to GAATTCAAAAGA (SEQ ID NO:10),
resulting in the introduction of an EcoRI site. The PCR reaction
conditions (five tubes in all) were: 20 cycles of 94.degree. C. for
30 seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1
minute; followed by a 4.degree. C. soak. The five samples were
combined into one tube and precipitated with 2 volumes of 100%
ethanol. The resulting pellet was resuspended in 10 .mu.l of water.
The sample was serially diluted into TE (10 mM Tris, 2 mM EDTA) as
1:5, 1:25, and 1:125 dilutions. DNA concentration was estimated by
running the PCR product on a 1% agarose gel. The expected
approximately 1 kb fragment was seen. The remaining 8 .mu.l of
product was used for recombination as described below.
[0059] An expression plasmid named pTAP96, containing the P.
methanolica GAP2 promoter, S. Cerevisiae alpha factor pre-pro
sequence, and a cDNA encoding leptin with an amino-terminal Glu-Glu
affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA
82:7952-4, 1985), was constructed via homologous recombination
using portions of the plasmids pTAP37 and pCZR189. Plasmid pTAP37
comprises a modified P. methanolica GAP1 promoter, the P.
methanolica ADE2 selectable marker, the gene for ampicillin
resistance in E. coli, the S. cerevisiae URA3 selectable marker,
and the CEN-ARS of S. cerevisiae. pCZR189 comprises the S.
Cerevisiae alpha factor pre-pro sequence and the leptin coding
sequence. One hundred microliters of competent yeast cells (S.
cerevisiae) were combined with 7 .mu.l of a mixture containing
approximately 1 .mu.g of NotI-cut pCZR189, 1 ,.mu.g PCR product
containing the GAP2 promoter as described above, and 100 ng of
EcoRI-cut pTAP37 vector, and the mixture was transferred to a 0.2
cm electroporation cuvette. The yeast/DNA mixture was electropulsed
at 0.75 kV (5 kV/cm), infinite ohms, 25 .mu.F. To each cuvette was
added 600 .mu.l of 1.2 M sorbitol, and the yeast was then plated in
two 300-.mu.l aliquots onto two -URA D plates and incubated at
30.degree. C.
[0060] After about 48 hours, the Ura.sup.+ yeast transformants from
a single plate were resuspended in 1 ml H.sub.2O and spun briefly
to pellet the yeast cells. The cell pellet was resuspended in 1 ml
of lysis buffer (2% t-octylphenoxypolyethoxyethanol (Triton.RTM.
X-100), 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five
hundred microliters of the lysis mixture was added to a
microcentrifuge tube containing 300 .mu.l acid-washed glass beads
and 200 .mu.l phenol-chloroform, vortexed for 1 minute intervals
two or three times, followed by a 5 minute spin in a
microcentrifuge at maximum speed. Three hundred microliters of the
aqueous phase was transferred to a fresh tube, and the DNA was
precipitated with 600 82 l ethanol, followed by centrifugation for
10 minutes at 4.degree. C. The DNA pellet was resuspended in 100
.mu.l H.sub.2O.
[0061] Forty .mu.l of electrocompetent E. coli cells (MC1061;
Casadaban et al., J. Mol. Biol. 138, 179-207, 1980) were
transformed by electroporation with 1 .mu.l of the yeast DNA
preparation at 2.0 kV, 25 .mu.F, and 400 ohms. Following
electroporation, 0.6 ml SOC (2% Bacto.TM. Tryptone (Difco
Laboratories), 0.5% yeast extract (Difco Laboratories), 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM glucose) was
plated in one aliquot on LB+Amp plates (LB broth, 1.8% Bacto.TM.
Agar (Difco Laboratories), 100 mg/L Ampicillin).
[0062] Cells harboring the correct expression construct for the
GAP2 promoter driving synthesis of the alpha factor pre-pro/leptin
fusion were screened via PCR using the same primers used to
generate the GAP2 promoter. The PCR conditions were: 25 cycles of
94.degree. C. for 30 seconds, 55.degree. C. for 30 seconds, and
72.degree. C. for 1 minute; followed by a 4.degree. C. soak. Two
positive clones were identified on a 1% agarose gel and were
subjected to sequence analysis. One of the correct clones was
selected and designated pTAP96.
[0063] Plasmid pTAP96 DNA was prepared by anion exchange
chromatography using a commercially available plasmid isolation kit
(QIAGEN.RTM. Plasmid Maxi Kit; Qiagen, Inc., Valencia, Calif.). DNA
was diagnostically cut with Scai, producing the expected bands of
approximately 1700 bp, 2250 bp doublet, and 6000 bp on a 1% gel. 1
.mu.g of pTAP96 DNA was then cut with NotI and transformed into
electrocompetent P. methanolica strain PMAD16 (disclosed in Example
4, below) as disclosed in U.S. Pat. No. 5,854,039. Transformants
were selected on -ADE DS plates (Table 1).
1 TABLE 1 -ADE DS 0.056%-Ade-Trp-Thr powder 0.67% yeast nitrogen
base without amino acids 2% D-glucose 0.5% 200X tryptophan,
threonine solution 18.22% D-sorbitol -Ade-Trp-Thr powder powder
made by combining 3.0 g arginine, 5.0 g aspartic acid, 2.0 g
histidine, 6.0 g isoleucine, 8.0 g leucine, 4.0 g lysine, 2.0 g
methionine, 6.0 g phenylalanine, 5.0 g serine, 5.0 g tyrosine, 4.0
g uracil, and 6.0 g valine (all L- amino acids) 200X tryptophan,
threonine solution 3.0% L-threonine, 0.8% L-tryptophan in H.sub.2O
For plates, add 1.8% Bacto .TM. agar (Difco Laboratories)
[0064] White colonies, indicating the presence of the ADE2 gene,
were patched onto -ADE plates, and cells were allowed to grow
overnight. The cells were then replica plated onto YEPD plates and
overlaid with a nitrocellulose membrane. The next day the filters
were washed gently under deionized H.sub.20, then denatured in
1.times. Western denaturing buffer (625 mM Tris, 625 mM glycine,
pH9.0, 5 mM .beta.-mercaptoethanol) at 65.degree. C. for 10
minutes. Filters were blocked for 30 minutes in TTBS (160 mM NaCl,
20 mM Tris pH7.4, 0.1% Tween 20) and 5% non-fat dry milk. The
filters were then exposed to an anti-Glu-Glu tag antibody
conjugated to horseradish peroxidase (5 .mu.l of antibody diluted
into 10 ml TTBS+5% non-fat dry milk) at room temperature for 1
hour. Filters were washed twice for 5 minutes in TTBS with no milk
and rinsed briefly in water. The filters were screened using
commercially available chemiluminescence reagents (ECL.TM. direct
labelling kit; Amersham Corp., Arlington Heights, Ill.) as a 1:1
dilution, and the filters were immediately exposed to film. One
clone produced a detectable signal.
Example 4
[0065] To generate a P. methanolica strain deficient for vacuolar
proteases, the PEP4 and PRB1 genes were identified and disrupted.
PEP4 and PRB1 sequences were amplified by PCR in reaction mixtures
containing 100 pmol of primer DNA, 1.times. buffer as supplied
(Boehringer Mannheim, Indianapolis, Ind.), 250 .mu.M dNTPs, 1-100
pmol of template DNA, and 1 unit of Taq polymerase in a reaction
volume of 100 .mu.l. The DNA was amplified over 30 cycles of
94.degree. C., 30 seconds; 50.degree. C., 60 seconds; and
72.degree. C., 60 seconds.
[0066] Using an alignment of PEP4 sequences derived from S.
cerevisiae (Ammerer et al., Mol. Cell. Biol. 6:2490-2499, 1986;
Woolford et al., Mol. Cell. Biol. 6:2500-2510, 1986) and P.
pastoris (Gleeson et al., U.S. Pat. No. 5,324,660), several sense
and antisense primers corresponding to conserved regions were
designed. One primer set, ZC9118 (SEQ ID NO:11) and ZC9464 (SEQ ID
NO:12) produced a PCR product of the expected size from genomic
DNA, and this set was used to identify a genomic clone
corresponding to the amplified region. DNA sequencing of a portion
of this genomic clone (shown in SEQ ID NO:13) revealed an open
reading frame encoding a polypeptide (SEQ ID No:14) with 70% amino
acid identity with proteinase A from S. cerevisiae.
[0067] Primers for the identification of P. methanolica PRB1 were
designed on the basis of alignments between the PRB1 genes of S.
Cerevisiae (Moehle et al., Mol. Cell. Biol. 7:4390-4399, 1987), P.
pastoris (Gleeson et al., U.S. Pat. No. 5,324,660), and
Kluyveromyces lactis (Fleer et al., WIPO Publication WO 94/00579).
One primer set, ZC9126 (SEQ ID NO:15) and ZC9741 (SEQ ID NO:16)
amplified a ca. 400 bp fragment from genomic DNA (SEQ ID NO:17).
This product was sequenced and found to encode a polypeptide (SEQ
ID NO:18) with 70% amino acid identity with proteinase B from S.
cerevisiae. The PRB primer set was then used to identify a genomic
clone encompassing the P. methanolica PRB1 gene.
[0068] Deletion mutations in the P. methanolica PEP4 and PRB1 genes
were generated using available restriction enzyme sites. The cloned
genes were restriction mapped. The pep4.DELTA. allele was created
by deleting a region of approximately 500 bp between BamnHI and
NcoI sites and including nucleotides 1 through 393 the sequence
shown in SEQ ID NO:13. The prb1.DELTA. allele was generated by
deleting a region of approximately 1 kbp between NcoI and EcoRV
sites and including the sequence shown in SEQ ID NO:17. The cloned
PEP4 and PRB1 genes were subcloned into pCZR139, a phagemid vector
(pBluescript.RTM. II KS(+), Stratagene, La Jolla, Calif.) that
carried a 2.4 kb Spel ADE2 insert, to create the deletions. In the
case of PEP4gene, the unique BamHI site in pCZR139 was eliminated
by digestion, fill-in, and religation. The vector was then
linearized by digestion with EcoRI and HindIII, and a ca. 4 kb
EcoRI - HindIII fragment spanning the PEP4 gene was ligated to the
linearized vector to produce plasmid pCZR142. A ca. 500 bp deletion
was then produced by digesting pCZR142 with BamHI and NcoI, filling
in the ends, and religating the DNA to produce plasmid pCZR143. The
PRB1 gene (.about.5 kb XhoI-BamHI fragment) was subcloned into
pCZR139, and an internal EcoRV-NcoI fragment, comprising the
sequence shown in SEQ ID NO:17, was deleted to produce plasmid
pCZR153.
[0069] Plasmid pCZR143 was linearized with Asp718, which cut at a
unique site. The linearized plasmid was introduced into the P.
methanolica PMAD11 strain (an ade2 mutant generated as disclosed in
U.S. Pat. No. 5,736,383). Transformants were grown on -ADE DS
(Table 1) to identify Ade.sup.+ transformants. Two classes of
white, Ade.sup.+ transformants were analyzed. One class arose
immediately on the primary transformation plate; the second became
evident as rapidly growing white papillae on the edges of unstable,
pink transformant colonies.
[0070] Southern blotting was used to identify transformants that
had undergone the desired homologous integration event. 100 .mu.l
of cell paste was scraped from a 24-48 hour YEPD plate and washed
in 1 ml water. Washed cells were resuspended in 400 .mu.l of
spheroplast buffer (1.2 M sorbitol, 10 mM Na citrate pH 7.5, 10 mM
EDTA, 10 mM DTT, 1 mg/ml zymolyase 100T) and incubated at
37.degree. C. for 10 minutes. Four hundred .mu.l of 1% SDS was
added, the cell suspension was mixed at room temperature until
clear, 300 .mu.l of 5 M potassium acetate was mixed in, and the
mixture was clarified by microcentrifugation for 5 minutes. 750
.mu.l of the clarified lysate was extracted with an equal volume of
phenol:chloroform:isoamyl alcohol (25:24:1), 600 .mu.l was
transferred to a fresh tube, 2 volumes of 100% ethanol was added,
and the DNA was precipitated by microcentrifugation for 15 minutes
at 4.degree. C. The pellet was resuspended in 50 .mu.l of TE (10 mM
Tris pH 8.0, 1 mM EDTA) containing 100 .mu.g/ml of RNAase A. Ten
.mu.l of DNA (approximately 100 ng) was digested in 100 .mu.l total
volume with appropriate enzymes, precipitated with 200 .mu.l
ethanol, and resuspended in 10 .mu.l of DNA loading dye. The DNA
was separated in 0.7% agarose gels and transferred to nylon
membranes (Nytran N.sup.+, Amersham Corp., Arlington Heights, Ill.)
in a semi-dry blotting apparatus (BioRad Laboratories, Richmond,
Calif.) as recommended by the manufacturer. Transferred DNA was
denatured, neutralized, and cross-linked to the membrane with UV
light using a Stratalinker (Stratagene, La Jolla, Calif.). To
identify strains with a tandem integration at PEP4, two probes were
used. One was a 1400 bp EcoRI-HindIII fragment from the 3' end of
PEP4. The second was a 2000 bp BamHI-EcoRI fragment from the 5' end
of PEP4. Fragments were detected using chemiluminescence reagents
(ECL.TM. direct labelling kit; Amersham Corp., Arlington Heights,
Ill.).
[0071] Parent strains harboring a tandem duplication of the
wild-type and deletion alleles of the gene were grown in YEPD broth
overnight to allow for the generation of looped-out, Ade.sup.-
strains. These cells were then plated at a density of 2000-5000
colonies per plate on adenine-limited YEPD plates, grown for 3 days
at 30.degree. C. and 3 days at room temperature. The shift to room
temperature enhanced pigmentation of rare, pink, Ade.sup.-
colonies. Loop-out strains were consistently detected at a
frequency of approximately one pink, Ade.sup.- colony per 10,000
colonies screened. These strains were screened for retention of the
wild-type or mutant genes by Southern blotting or by PCR using
primers that spanned the site of the deletion. An ade2-11
pep4.DELTA. strain was designated PMAD15.
[0072] The PRB1 gene was then deleted from PMAD15 essentially as
described above by transformation with plasmid pCZR153. Blots were
probed with PCR-generated probes for internal portions of the PRB1
and ADE2 genes. The PRB1 probe was generated by subcloning a 2.6 kb
ClaI-SpeI fragment of PRB1 into the phagemid vector
pBluescript.RTM. II KS(+) to produce pCZR150, and amplifying the
desired region by PCR using primers ZC447 (SEQ ID NO:19) and ZC976
(SEQ ID NO:20). The ADE2 probe was generated by amplifying the ADE2
gene in pCZR139 with primers ZC9079 (SEQ ID NO:21) and ZC9080 (SEQ
ID NO:22). The resulting ade2-11pep4.DELTA. prb1.DELTA. strain was
designated PMAD16.
[0073] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 0
0
* * * * *