U.S. patent application number 10/232614 was filed with the patent office on 2003-03-13 for expression vector for improved production of polypeptides in yeast.
This patent application is currently assigned to Patentpharm AG.. Invention is credited to Schreier, Thomas, Voegeli, Rainer.
Application Number | 20030049785 10/232614 |
Document ID | / |
Family ID | 8166735 |
Filed Date | 2003-03-13 |
United States Patent
Application |
20030049785 |
Kind Code |
A1 |
Schreier, Thomas ; et
al. |
March 13, 2003 |
Expression vector for improved production of polypeptides in
yeast
Abstract
A new expression vector for the production of a polypeptide in
yeast. The vector includes a sequence coding for the polypeptide
and other sequences allowing expression of the polypeptide only in
yeast. The other sequences lack any non-yeast sequences. Other
embodiments include a yeast strain comprising such a vector, a
method for the production of the vector, a method for the
production of the yeast strain by transformation of a yeast strain
with the new vector, and a method for the production of a
polypeptide in the transformed yeast strain by fermentation thereof
followed by isolation of the polypeptide.
Inventors: |
Schreier, Thomas;
(Bubendorf, CH) ; Voegeli, Rainer; (Bubendorf,
CH) |
Correspondence
Address: |
PENNIE & EDMONDS LLP
1667 K STREET NW
SUITE 1000
WASHINGTON
DC
20006
|
Assignee: |
Patentpharm AG.
|
Family ID: |
8166735 |
Appl. No.: |
10/232614 |
Filed: |
September 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10232614 |
Sep 3, 2002 |
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09518658 |
Mar 3, 2000 |
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6451559 |
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09518658 |
Mar 3, 2000 |
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PCT/EP97/04289 |
Sep 5, 1997 |
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Current U.S.
Class: |
435/69.1 ;
435/254.2; 435/320.1; 435/483; 435/91.2; 536/23.7 |
Current CPC
Class: |
C12N 9/0089 20130101;
C12N 15/81 20130101 |
Class at
Publication: |
435/69.1 ;
435/91.2; 435/254.2; 435/483; 435/320.1; 536/23.7 |
International
Class: |
C12P 021/02; C07H
021/04; C12N 001/18; C12P 019/34; C12N 015/74 |
Claims
What is claimed is:
1. An isolated expression vector construct for the production of a
polypeptide in yeast, the expression vector comprising (1) a DNA
sequence encoding for the polypeptide and (2) DNA sequences
allowing for the expression of the polypeptide in yeast, wherein
the expression vector, except for any DNA sequences encoding for
non-yeast polypeptides, lacks any non-yeast DNA sequences, the
expression vector being obtainable by a process comprising: (A)
isolating genomic DNA from a yeast; (B) isolating a chromosomal
fragment carrying a Cu/Zn SOD gene with upstream and downstream
regions; (C) subcloning said chromosomal fragment into a plasmid
pCRII; (D) excising and purifying a fragment carrying the Cu/Zn SOD
gene using restriction enzymes BamHI and SalI; (E) subcloning the
purified fragment into the BamHI-SalI sites of vector pEMBLyex4 to
produce the vector pEMBL-SOD 374 with the Cu/Zn SOD gene and an
upstream sequence; (F) deleting the polylinker multiple cloning
site from a sample of vector pEMBL-SOD 374 by double digestion of
the polylinker with the enzymes SstI and HindIII; (G) isolating a
HindIII-SacI fragment with the yeast and bacterial sequences of the
vector pEMBL without the polylinker; (H) purifying and
concentrating the HindIII-SacI fragment; (I) double digestion of a
sample clone pEMBL-SOD 374 carrying the Cu/Zn SOD gene with enzymes
EcoRI and SalI to isolate a DNA fragment carrying the entire open
reading frame of the Cu/Zn SOD gene together with upstream and
downstream sequences; (J) producing blunt ends on the EcoRI-SalI
fragment; (K) subcloning the EcoRI-SalI fragment into the
HindIII-SacI fragment; (L) isolating clones from the ligation by
random screening that have the vector pEMBL-SOD without the
multiple cloning site sequences; (M) digesting pEMBL-SOD without
multiple cloning site sequences with StuI and NruI; (N) isolating
the remaining the fragment carrying only yeast sequences; and (O)
ligating the ends of the fragment to produce vector pEMBL-SOD.
2. The expression vector of claim 1, wherein the yeast is
Saccharomcyces cerevisiae.
3. A method for production of an expression vector, the method
comprising: isolating a first chromosomal fragment from a yeast,
wherein the fragment comprises a Cu/Zn SOD gene with upstream and
downstream regions; subcloning the first fragment into a plasmid
pCRII; excising a second fragment carrying the Cu/Zn SOD gene from
the plasmid pCRII carrying the first fragment; subcloning the
second fragment into the BamI-SalI sites of a vector pEMBLyex4 to
obtain a vector pEMBL-SOD 374; isolating a Hind III-Sac I fragment
from the vector pEMBL-SOD 374, wherein the Hind III-Sac I fragment
comprises yeast and bacterial sequences of the vector pEMBLyex4 but
not does not include the polylinker multiple cloning site;
isolating an EcoRI-SalI fragment from a sample of clone pEMBL-SOD
374, wherein the EcoRI-SalI fragment comprises the entire open
reading frame of the Cu/Zn SOD gene and at least a portion of the
upstream and downstream sequences; subcloning the EcoRI-Sal I
fragment into the Hind III-Sac I fragment to obtain a third
fragment; isolating clones of the third fragment, wherein the
isolated clones lack the multiple cloning site sequences; digesting
an isolated clone to delete all non-yeast bacterial sequences to
obtain a fourth fragment comprising the marker Leu-2d and the Cu/Zn
SOD gene; and rejoining ends of the fourth fragment to produce a
vector comprising the Cu/Zn SOD gene, the Leu-2d marker, the origin
of replication of the two-micron vector, and the entire yeast
expression hybrid promoter cassette UAS GAL/CYC, wherein the vector
lacks bacterial sequences and the multiple cloning site.
4. The method of claim 3, wherein the yeast is Saccharomcyces
cerevisiae.
5. The method of claim 4, further comprising transforming a yeast
strain with the expression vector that lacks, except for any DNA
sequences coding for non-yeast polypeptides, any non-yeast DNA
sequences.
6. The method of claim 5, wherein the yeast strain is
Saccharomcyces cerevisiae.
7. The method of claim 6, wherein the strain is GRF18.
8. The method of claim 3, wherein the vector lacks, except for any
DNA sequences coding for non-yeast polypeptides, any non-yeast DNA
sequences.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 09/518,658, filed Mar. 3, 2000, which
application is a continuation of the U.S. phase of co-pending
International Application No. PCT/EP/04289, filed Sep. 5, 1997.
Each of the prior applications are incorporated herein in their
entireties by reference.
FIELD OF THE INVENTION
[0002] The invention relates to a new expression vector for the
production of a polypeptide in yeast, a yeast strain being
transformed with such vector, and methods for the production of the
vector, yeast strain and polypeptide.
BACKGROUND OF THE INVENTION
[0003] Genetic engineering techniques for expression in yeasts
commonly use shuttle vectors. The shuttle vectors have nucleotide
sequences coding for a particular polypeptide combined with
sequences necessary for expression in yeast, such as a yeast
promoter. These shuttle vectors also have additional sequences that
allow for expression in bacteria, such as Escherichia coli, or
other microorganisms. Such additional non-yeast sequences are
useful only for the construction of the vectors. However, they are
superfluous for the expression in yeast. In fact they may hinder
the efficient expression of the polypeptide in yeast or retard the
replication of the organism because the superfluous nucleotides
must also be doubled, which is an energy consuming process.
[0004] The yeast Saccharomyces cerevisiae is usually an excellent
microorganism for the production of both homologous and
heterologous proteins. This is because of its well characterized
genetic system, rapid growth, and technical advantages of
manipulation. Additionally, the development of DNA transformation
systems for the introduction of cloned genes and their inexpensive
and safe overproduction in simple fermentation conditions, has made
this organism particularly useful for large-scale industrial
practice.
[0005] A number of yeast polypeptides are known in the art. Of
particular interest are the superoxide dismutases. The yeast
Saccharomyces cerevisiae contains two species of superoxide
dismutases (EC 1.15.11), the copper/zinc-(Cu/Zn SOD) and the
manganese-(Mn SOD) containing forms. The Cu/Zn SOD is localized in
the cytoplasm while the manganese enzyme is restricted to the
mitochondrial matrix. This enzyme is assumed to provide in vivo
protection against toxic free radicals generated within cells as
intermediates of normal metabolism (Bilinski, T. et al. Biochem.
Biophys. Res. Commun. 130: 533-539 (1985), Van Loon A. P. G. M. et
al. Proc. Natl. Acad. Sci. USA 83: 3820-3824 (1986), Lee F. J. et
al. J. Free Rad. Biol. Med. 1: 3 19-325 (1985), Galiazzo F. et al.
Biochim. Biophys. Acta 965: 46-51 (1988)). Consequently, it is
expected to be useful for preventing or treating potential damage
in human, particularly damage from cell aging and senescence (Rosen
D. R. et al. Nature 362, 59-62 (1993), McCord J. M. and Fridovich
I. J. Biochem 244: 6049-6055 (1969),. McCord J. M. et al. Proc.
Natl. Acad. Sci. USA 68: 1024-1027 (1971), McCord J. M. N. Engl. J.
Med. 312: 159-163 (1985)).
[0006] The Cu/Zn SOD gene from Saccharomyces cerevisiae was cloned,
sequenced (Bermingham-McDonogh 0., et al. Proc. Nat. Acad. Sci. USA
85: 4789-4793 (1988)), and the structure and mechanism of action of
the enzyme is well characterized (Djinovic K. et al. J. Mol. Biol.
225: 791-809 (1992), O'Neill P. et al. Biochem. J. 251: 41-46
(1988)). The Cu/Zn SOD is an abundant metalloenzyme present in the
cytoplasm of most aerobic and many anaerobic organisms, whose
activity catalyzes the dismutation of the superoxide anion to
dioxygen and hydrogen peroxide.
[0007] It is an object of the present invention to improve on the
yields of polypeptides in the fermentation processes of yeasts
transformed with expression vectors coding for such polypeptides.
It is a further object to provide new vectors which are able to
express desired polypeptides in yeast in larger amounts as compared
to previous processes. A further object is to provide new yeast
strains transformed with such vectors that are superior compared to
the wild-type strain or those which are transformed with shuttle
vectors.
SUMMARY OF THE INVENTION
[0008] The present invention provides for expression vectors and a
yeast strains. In particular a Saccharomyces cerevisiae strain
transformed with a vector, which produces higher levels of yeast or
non-yeast polypeptides compared to the wild-type strain or those
transformed with a shuttle vector. Methods for the preparation of
such expression vector, yeast strains and endogenous yeast
polypeptides are set forth.
[0009] Numerous aspects and advantages of the invention will be
apparent to those skilled in the art upon consideration of the
detailed description and the drawings of the invention which
provides illustrations of the practice of the invention in its
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1. Depiction of the coding region for the Cu/Zn SOD
gene from yeast strain S288C with the positions of the external
primers SOD3 and SOD2 and the internal primer SOD4 designated in
the upstream and downstream regions of the SOD gene locus.
[0011] FIG. 2. Depiction of the plasmid construct pEMBL-SOD 374,
derived from pEMBLyex4, with a 374 bp upstream region, the coding
sequence and a downstream region of the yeast Cu/Zn SOD gene under
the control of the yeast GAL/CYC promoter.
[0012] FIG. 3. Depiction of a map of the final plasmid pEMEL-SOD
without sequences from multiple cloning sites or Esherichia coli
comprising a 374 upstream region, the coding sequence and a
downstream region of the yeast Cu/Zn SOD gene between the
restriction sites EcoRI and HindIII, under the control of the yeast
GAL/CYC promoter, it is comprised of only yeast sequences and
replicates only in yeast.
DETAILED DESCRIPTION OF THE INVENTION
[0013] In the present invention the following primer DNA sequences
have been used, the structures of which are precisely shown in the
Sequence Identification Listing (the bp regions of the primers were
taken from the EMBL vector, GeneBank accession No. J03279):
[0014] SEQ ID NO 1: is the external primer SOD-3, upstream region
81-102 bp
[0015] SEQ ID NO 2: is the external primer SOD-2, downstream region
10 18-1036 bp
[0016] SEQ ID NO 3: is the internal primer SOD-4, region 97 1-991
bp
[0017] A preferred embodiment of the present invention is a new
expression vector for producing polypeptides in yeast comprising
the coding sequence for said polypeptide, and additional sequences
that allow for expressing the polypeptide in yeast, these
additional sequences lack any non-yeast sequences.
[0018] The term "expression vector" is intended mean a vector, in
particular a DNA vector, such as a plasmid, which comprises a
sequence coding for a polypeptide, a promoter sequence in reading
frame with the coding sequence, and optionally other sequences,
which are needed for efficiently producing or using the vector,
such as an origin of replication (ori), a leader sequence, a
terminator and a selection marker. Such optional other sequences
are only derived from yeasts and are well known in the art.
[0019] The sequence coding for said polypeptide may be a yeast or
non-yeast sequence. Yeast sequences may code for yeast polypeptides
with enzyme functions. Examples of Yeast enzymes include
antioxidative enzymes like superoxide dismutase (SOD), thiol
specific antioxidant (TSA), and cytochrome c peroxidase, proteases
like cerevisin precursor PRB 1, proteinase inhibitors including
proteinase B inhibitor 2, cytokines, and various others.
[0020] Sequences coding for non-yeast polypeptides may be derived
from any living organism, particularly humans and animals. Such
polypeptides are preferably useful in the medical arts and include
but are not limited to human insulin, tissue plasminogen activator,
interferons, erythropoietin, growth factors like keratinocyte
growth factor, tryptase, Protein C activator, tissue inhibitors of
metalloproteinases (TIMP's), elastase inhibitors, and various
others. The sequences coding for such useful polypeptides are known
in the art.
[0021] All these sequences are under the control of yeast
promoters. Useful yeast promoters include the GAL/CYC promoter for
example and are known in the art.
[0022] The final vector of the invention is only replicable in
yeast cells. With the exception of the non-yeast sequence coding
for any desired non-yeast polypeptide, the vector lacks any
non-yeast sequences.
[0023] A preferred vector according to the invention is a yeast
plasmid comprising the Cu/Zn SOD gene which is under the control of
the GAL/CYC promoter, and is in particular the plasmid named
pEMBL-SOD, without multiple cloning site or Escherichia coli
sequences.
[0024] This plasmid may be used as a starting plasmid for
constructing an expression vector where the Cu/Zn SOD gene is
exchanged for sequences coding for other polypeptides.
[0025] In a further embodiment the invention provides a method for
the production of the new expression vector defined hereinbefore.
The method for producing the new expression vector of the invention
is characterized by the excision of any non-yeast sequences from a
shuttle vector able to express a polypeptide in a yeast strain.
Optionally, the sequence coding for said polypeptide is replaced by
a sequence coding for another polypeptide.
[0026] The new expression vectors are obtained by conventional
techniques from known shuttle vectors, such as yeast integration
plasmid YIp, yeast replication plasmid YRp, yeast centromeric
plasmid YCp, the yeast episomal plasmid YEp. These vectors comprise
a polypeptide gene and lack any non-yeast DNA sequences.
[0027] The starting shuttle vectors may already have the sequence
coding for the desired polypeptide under the control of any yeast
promoter, like the GAL/CYC promoter. Such vectors are for example
the plasmid pEMBL-SOD 374 or pEMBL-SOD ATG. These plasmids are used
as intermediates for the production of the final vector according
to the invention. If the gene is not yet available, constructing
the vector starts with the isolating the gene coding for the
desired polypeptide from a known source, e. g., from a human or
animal or a wild-type microorganism strain. The gene is multiplied
through PCR with synthetic primers, and inserted into the vector,
usually a shuttle vector. Using restriction enzymes, all non-yeast
sequences including but not limited to bacterial sequences,
multiple cloning sites, bacterial origins of replication (ORIs),
selectable markers, the origin of replication of the filamentous
bacteriophage fl, the ampicillin resistant gene, and the like are
deleted from the intermediate vectors.
[0028] The following is a more detailed discussion of the method
used: Gene expression requires placing a gene, coding for a
polypeptide of interest, under the control of a strong yeast
promoter that directs synthesis of the corresponding messenger RNA.
The DNA regulatory elements required for expression are carried by
yeast vectors.
[0029] These vectors are shuttle vectors that may be propagated in
yeast strains as well as in the bacterium Escherichia coli for
convenient manipulations and large scale preparations of the
different intermediate plasmids.
[0030] A number of different yeast integrating (Yip), replicating
(YRp), centromere (YCp) and episomal (YEp) plasmid vectors have
been developed (Rose A. B., Broach J. R. Methods in Enzymology,
185:234-279 (1990), Schneider J. C., and Guarente L. Methods in
Enzymology, 194: 373-388 (1991)).
[0031] The plasmid that was chosen for the expression of the Cu/Zn
SOD gene is the specific YEp (yeast episomal plasmid) shuttle
vector pEMBLyex4 of 8.800 base pairs (Cesarani and Murray, in
Setlow J. K. (ed) Genetic Engineering: Principle and Methods,
Volume 9, Plenum Press, NY 134-135 (1987)).
[0032] Such a vector carries the 2-micron yeast episome (a small
double-stranded DNA plasmid present in the nuclei of most
Saccharomyces cerevisiae strains) which provides high mitotic
stability and the ability to be autonomously replicated (Murray, J.
A. H., Mol. Microbiol., 1: 1-4 (1987), Hartley and Donelson,
Nature, 286: 860-864 (1980), Clark-Walker G. D., and Miklos G. L.
G., Eur. J. Biochem., 41: 359-365 (1974), Futcher A. B., and Cox B.
S., J. Bacteriol., 157: 283-290 (1984)).
[0033] The persistence of the plasmid is due to the presence, in
the 2-micron moiety, of the REP 3 locus (for the partitioning of
the plasmid during cell division) and the ARS sequence (origin of
replication).
[0034] The plasmid pEMBLyex4 carries the LEU 2 and URA 3 selectable
markers which are extremely useful both to select the initial yeast
cell transformants and to provide constant pressure to maintain the
plasmid in the yeast cell (Alani E. et al., Genetics, 116: 541
(1987), Gritz L., and Davies J., Gene, 25: 179 (1983), Kaster K.
R., et al., Curr. Genet., 8: 353 (1984), Rine J., et al., Proc.
Natl. Acad. Sci. USA, 80:6750 (1983)).
[0035] In general, however, these kinds of plasmids achieve a good
maintenance even in the absence of positive selection. In such a
situation, cells can lose the plasmid at a rate of about 4 percent
per generation. The pEMBLyex4 forms part of a special class of
2-micron vectors with a very high copy number (about 100-200 per
cell).
[0036] Moreover, yeast strains lacking the 2-micron episome
(cir.degree.) to propagate the plasmid were used. The stability of
pEMBLyex4 in such strains is known to be very high even without
continued selection pressure. The pEMBLyex4 plasmid includes the
entire yeast expression hybrid cassette UAS GAL/CYC. The promoter
cassette contains an upstream activation site (UAS sequence) and
the promoter region (TATA box) for both high levels of
transcription of the downstream gene and regulation of expression.
The pEMBLyex4 plasmid also includes a multiple cloning site (MCS)
for inserting the gene and a termination region (Guarente L. et
al., Proc. NatI. Acad. Sci., USA, 79: 7410-7414 (1982)). The hybrid
cassette UAS GAL/CYC has from 5' to 3' the following regions:
[0037] a 365 bp fragment (Sau3A-XhoI) from the upstream activation
sequence of the region between the yeast GAL4 and GAL 10 genes
which contains the binding region for the GAL4 product;
[0038] a 250 bp region (XhoI-SstI) containing the promoter of the
yeast gene CYC1, which carries the TATA box and the mRNA start
sites but without the ATG region;
[0039] a polylinker (SstI-HindlII) of 95 bp with unique restriction
enzyme sites;
[0040] a 250 bp region (in a HindIII-SnaBI fragment) carrying
polyadenylation and transcription terminator signals, from the
2-micron FLP gene.
[0041] The expression system is regulated by the GAL4 and GAL8O
gene products. The GAL4 protein is a transcriptional activator that
binds to the UAS gal sequences. The activity of the GAL4 protein is
inhibited by the binding of the GAL8O protein to its
carboxy-terminal region. The system is repressed by glucose, which
inhibits the binding of GAL4 protein to the UASgal, and is induced
by galactose, which causes the dissociation of the GAL8O protein
from the GAL4 protein.
[0042] In a further embodiment the invention provides a novel yeast
strain transformed with an expression vector according to the
invention.
[0043] Yeast species are any known species useful for the
expression of yeast or non-yeast polypeptides, for example
Saccharomyces cerevisiae or Saccharomyces occidentalis, or non
Saccharomyces yeast species, e. g., Hansenula polymorpha, Pichia
pastoris, Schwanniomyces occidentalis, and Pichia stipitis.
[0044] A preferred novel yeast strain is, for example Saccharomyces
cerevisiae, with improved ability to synthesize the Cu/Zn SOD
enzyme through insertion of the relative homologous gene in the
intracellular compartment.
[0045] Preferred intermediate yeast strains according to the
present invention are for example GRF 18 transformed with plasmid
pEMBL-SOD 374 or plasmid pEMBL-SOD ATG which have been produced,
isolated and characterized.
[0046] A further object of the invention is a method for the
production of a yeast strain transformed with an expression vector
coding for an endogenous yeast polypeptide, lacking any non-yeast
sequences. This yeast strain is able to overproduce said yeast
polypeptide and is characterized in that it it is transformed with
a new vector described hereinbefore.
[0047] The transformation follows methods common in the art, such
as the LiCl method of Ito et al., as modified by R. H. Schiestl et
al. (1989), Current Genetics, 16, 339-346, or the method of Hinnen
et al., (1978), Proc. Natl. Acad. Sci. USA, 75, 1929-1933.
[0048] In a further embodiment, the invention provides a method for
the production of a polypeptide in a yeast strain comprising
fermentation of a yeast strain transformed with an expression
vector according to the invention.
[0049] Fermentation follows methods common in the art, such as the
fed-batch method with a controlled fed of glucose during the growth
phase and induction of expression by addition of galactose in the
middle of the growth phase according to Alberghina, L., et al.,
(1991), Biotech. and Appl. Biochem., 14, 82-92.
EXAMPLES
[0050] The following examples are presented by way of illustration
of the invention and are directed to procedures carried out for the
isolation and characterization of a yeast enzyme gene: the Cu/Zn
SOD gene from traditional strains of S. Cerevisiae. Examples are
provided for procedures for expressing yeast enzymes encoded by
their genes in yeast strains under the control of a strong yeast
promoter, and to the development and characterization of a yeast
strain able to produce high levels of the enzyme; the SOD protein
is used as an example.
[0051] Abbreviations
[0052] Hereinbefore and hereinafter the following abbreviations are
used:
1 ARS Autonomously Replicating Sequence EDTA ethylenediamine tetra
acetic acid EMBL European Molecular Biology Laboratory LB Luria
Bertani Medium MCS multiple cloning site PCR polymerase chain
reaction PIU Pyrogallol Inhibitory Units REP Replikon (short
DNA-sequence which serves in cells as origin of DNA replication)
SDS sodium dodecyl sulfate SOD superoxide dismutase TBE
Tris-Borate-EDTA buffer TE Tris-EDTA buffer w/oColi without
Eseherichia coli sequences w/oMCS without multiple cloning site YCp
yeast centromeric plasmid YEp yeast episomal plasmid YEPD Yeast
Extract-Peptone-Dextrose medium YIp yeast integration plasmid Yrp
yeast replication plasmid
Example 1
[0053] Extraction and Purification or Yeast Genomic DNA
[0054] This example relates to the extraction and purification of
yeast genomic DNA to be used for the isolation of the yeast Cu/Zn
SOD gene.
[0055] The DNA source to used to isolate the Cu/Zn SOD gene can be
any wild-type yeast strain. In this specific case DNA was extracted
from Saccharomyces cerevisiae strains S288C wild-type, gal2, and
W309 wild-type.
[0056] Similar DNA extraction could be performed by using other
wild-type yeast species a DNA source, for example W303.
[0057] The haploid yeast strain S288C is a typical strain that is
currently used in most of the Molecular Biology laboratories around
the world for the isolation of yeast genes, and genetic and
biochemical studies (Mortimer R. K. and Johnson J. R., Genetics,
113:13 (1986)). The strain W309 has been considered as a potential
alternative source. Both strains are known to carry a copy of the
wild-type Cu/Zn SOD gene in their genome. The strains were provided
by The Departement of Physiology and General Biochemistry,
University of Milan.
[0058] A modified protocol for extracting total yeast genomic DNA
according to methods of Cryer, Ecclesial and Marmur, Methods Cell
Biology, 12:39-44 (1975) was used as follows.
[0059] Yeast cells, from a petri plate with YEPD medium, were
inoculated in 200 mL of complete medium YEPD (1% Bacto-yeast
extract, 2% Bacto-peptone, 2% Dextrose) and grown in a 1 liter
flask, overnight with shaking at 300.degree. C., until late
exponential phase (about 8.times.10.sup.7 cells/mL).
[0060] A total of 8.times.10.sup.8 cells were used for the
extraction of the DNA. 10 mL of cells were spun down and
concentrated in a polypropylene tube and the pellet was transfered
to a 1.5 mL Eppendorf tube. 300 microliters of lysis buffer (NaCl
0.15 M, EDTA 0.1 M pH 8, SDS 1%) and 300 microliters of glass
microbeads (diameter of 0.5 mm) were added to the Eppendorf tube.
The cells were vortexed five times on ice, with pauses of 1
min.
[0061] The cell suspension was homogenized by vortexing with 600
microliters of phenol-chloroform-TE solution and spun for 2 min.
The upper aqueous phase was transferred to a new Eppendorf tube and
600 microliters of chloroform-isoamylic-alcohol solution (24:1) was
added and mixed.
[0062] The upper phase was transfered to a new tube and incubated
at 37.degree. C. for 30 min with RNAase at a final concentration of
1 mg/mL. After ethanol precipitation, the pellet was resuspended in
TE buffer (Tris 10 mM, EDTA 1 mM, pH 8).
[0063] About 100 mg of yeast genomic DNA, at about 1 mg/mL, was
obtained from each preparation following this method. This was
enough for many experiments.
Example 2
[0064] Isolating the Chromosomal Region Carrying the Cu/Zn SOD
Gene
[0065] Polymerase Chain Reaction (PCR) was used to isolate the
chromosomal region carrying the Cu/Zn SOD gene.
[0066] The Cu/Zn SOD gene maps to the right arm of chromosome X in
S. Cerevisiae between the cyc1-rad6-SUP4-cdc8 cluster and cdc11
region (Chang et al. J. Biol. Chem. 266: 44 17-4424 (1991). It has
no introns in its coding sequence. Consequently, it is possible to
isolate the entire translated region directly from genomic DNA by
PCR. In addition, both the 5' upstream region and the 3' downstream
region of the gene are known.
[0067] To perform the PCR reaction, pairs of synthetic
oligonucleotide primers which span the SOD gene between the
upstream and the downstream region are needed.
[0068] The primers for the PCR were designed using the OLIGO primer
analysis software, Version 4.0 (National Biosciences Inc. Plymouth)
to cover the region of the published Cu/Zn SOD gene sequence of
1037 bases in Berminghan-McDonogh O., Gralla E., Valentine J.,
Proc. Natl. Acad. Sci. USA 85:4789 (1988) (EMBL/Gene Bank,
Accession No. J03279).
[0069] The primers were synthesized on an Applied Biosystem 392
Nucleic Acid synthesizer (Perkin-Elmer Corp., Foster City, Calif.)
and purified by gel filtration with Sephadex G-25 DNA grade NAP-25
Columns (Pharmacia P-L Biochemicals Inc; Milwaukee, Wis.).
[0070] As a general strategy aimed to increase the chance for
isolating the Cu/Zn-SOD gene from the yeast genome the so called
"semi-nested PCR" was used. This strategy uses a two-step protocol
requiring three different primers (FIG. 1).
[0071] The first step, which uses two primers (one upstream and one
downstream primer, SOD-3 and SOD-2), allows for the amplification
of a larger region of the genome. This step is followed by a second
amplification, which uses a new internal primer (SOD-4) and one of
the previous two (SOD-3), which finally permits the isolation and
recovery of the SOD gene.
[0072] The first round of PCR amplification was performed using the
following primers: SOD-3 (upstream primer, region 81-102 of the
sequence entered in EMBL/Gene Bank, Accession No. J03279):
[0073] SEQ. ID NO: 1 5'-GGA CGT AAG CAT CTC TGA AGT G-3' (22 mer,
T.sub.M=66.degree. C.),
[0074] SOD-2 (downstream primer, region 1018-1036 of the sequence
entered in EMBL/Gene Bank, Accession No. J03279),
[0075] SEQ. ID NO:2 5'-GCC GTC GAC GGA CCC CTC AAG ACC CCT C-3' (28
mer, T.sub.M=64.degree. C.).
[0076] The SOD-2 primer has a matching region of 19 bases of length
(T.sub.M=64.degree. C.) and a 5'-non-matching region which carries
a BamHI restriction site.
[0077] DNA amplification was performed in 50 mM KCl, 10 mM Tris-HCl
pH 8.3, 1.5 mM MgCl.sub.2, 500 mM of each deoxynucleotide (dATP,
dCTP, dGTP, dTTP), 0.5 mM of each primer, 50 or 100 ng of genomic
DNA and 2.5 Units of Taq DNA polymerase (Perkin-Elmer Corp.) in an
100 mL reaction volume. Times and temperatures used in each
amplification stage were as follows: 1 min at 94.degree. C. for the
denaturation, 1.5 min at 63.degree. C. for the annealing and 2 min
for the elongation.
[0078] The PCR reaction generated a DNA fragment of 965 base pairs
(bp) in length, as seen in agarose gel electrophoresis, which may
include the entire coding sequence of the SOD gene of 462 bp, an
upstream sequence of 331 bp and a downstream sequence of 172
bp.
[0079] The second round (semi-nested PCR) was performed using the
primer SOD-3 and a third internal primer SOD-4 which spans the
region 971-991 of the sequence entered in EMBL/Gene Bank, accession
No. J03279 and has the following sequence:
[0080] SEQ ID NO:3 5'-GCC GTC GAC ACA CTT GGT GAA TGA TCA
AGG-3'.
[0081] Primer SOD-4 has a matching region of 21 bases of length
(T.sub.M=60.degree. C.) and a 5' non-matching region which carries
a SalI restriction site.
[0082] The semi-nested PCR reaction, performed with the above
conditions except for the annealing temperature of 60.degree. C.,
generated a shorter DNA fragment of 920 base pairs (bp) of length
which may include the entire coding sequence of the SOD gene of 462
bp, an upstream sequence of 331 bp, and a downstream sequence of
127 bp.
Example 3
[0083] Subcloning of the Cu/Zn SOD Gene into the Plasmid Vector
pCRII
[0084] This example relates to the subcloning of the Cu/Zn SOD gene
into the plasmid vector pCRII.
[0085] Following amplification, the products of the reactions were
loaded on a 1.2% low-melting temperature agarose gel and run on an
electrophoresis apparatus (MiniSubgel DNA cell, BIO-RAD
Laboratories, Inc. Hercules, Calif., USA,). The appropriate DNA
band was then isolated from the agarose gel through standard
methods and purified by phenol extraction (Sambrook J., Fritsch E.
F., and Maniatis T. Molecular Cloning. A manual laboratory, Cold
Spring Harbor Laboratory, New York. 1989).
[0086] Finally, the purified DNA fragment was inserted in the
multiple cloning site (MCS) of the linearized plasmid pCRII of 3932
bp of length using the TA Cloning System (Invitrogen Corp. San
Diego, Calif.), by ligation with T4 DNA ligase, at 12.degree. C.
for 16 h.
[0087] The pCRII vector is a cloning vector which contains single
3' deoxythymidylate overhangs that allows for direct ligation of
PCR products, and both ampicillin and kanamycin resistence genes
for selection in E. coli cells.
[0088] The construct (plasmid pCRII plus the insert) was then
transfected and replicated in E. coli cells HB101.
[0089] Finally, the plasmid DNA was extracted from bacterial cells
by the alkaline lysis method of Birnboim and Doly (Birnboim H. C.,
and Doly. J. (1979) Nucleic Acids Research, 7:1513) and purified by
Nucleobond AX-100 cartridges (Macherey-Nagel GmbH, Duren,
Germany).
Example 4
[0090] Construction of Plasmid pEMBL-SOD 374
[0091] The construct was prepared by using the pEMBLyex4 plasmid as
a vector in which the previously isolated locus containing the SOD
gene, the downstream, and the modified upstream regions were
cloned.
[0092] The construct was named pEMBL-SOD 374 and is shown in FIG.
2. It is a construct in which the subcloned fragment carries the
coding region of the SOD gene (462 bp) and an upstream region of
374 bp.
[0093] To produce such a construct, the fragment which carries the
Cu/Zn SOD gene, previously subcloned in pCRII plasmid, was directly
excised by the restriction enzymes BamHI and SallI. This enzymatic
digestion produced a BamHI-SalI 963 bp fragment which was purified
by gel electrophoresis and subcloned into the BamHI-SalI sites of
the vector pEMBLyex4.
Example 5
[0094] Preparation of Yeast Strains X4004 and GRF18 Transformed
with pEMBL-SOD 374
[0095] Expression of the yeast Cu/Zn-SOD gene from the plasmid
pEMBL-SOD 374 was tested following its insertion in two different
strains of S. cerevisiae typically utilized for the expression of
homologous or heterologous genes in yeasts (Martegani et al., Appl.
Microbiol. Biotechnology, 37:604-608 (1992), Alberghina, L. et al.,
Biotechnology and Applied Biochemistry, 14:82-92 (1991), Pradyumna
K. et al., Biotechnology and Bioeng. 40:235-246 (1992), Yong Soo
Park et al., Biotechnology and Bioeng., 41:854-861 (1993), Scott et
al., Biotechnology and Bioeng., 41:801-810 (1993), Jih-Han Hsieh et
al., Biotechnology and Bioeng., 32:334-340 (1988)).
[0096] The following haploid strains were used:
[0097] X4004, whose genotype is: MATa/lys5/ura3/met2/trp1; and
[0098] GRE18, whose genotype is: MATa/leu2-3,112/His3-11,15.
[0099] These strains can be fermented at high biomass quite
efficiently in selective or semisynthetic media due to the markers
present on the plasmid pEMBLyex4 (LEU2-d and URA3) which
complements, respectively, the leucine auxotrophy in the leu2-GRF18
strain (the GRF18 strain carries a leu 2-3,112 double frameshift
mutation that reverts extremely rarely) and the uracil auxotrophy
of the ura3-X4004 strain.
[0100] Naturally, following insertion of the plasmid, the novel
strains loses those specific auxotrophies. GRF 18, carrying the
novel plasmid will still be auxotroph for histidine, while X4004,
carrying the novel plasmid will still be auxotroph for methionine
and tryptophane.
[0101] The yeast transformation with the plasmid constructs were
performed as follows. Before transformation:
[0102] yeast strains (X4004 or GRF18) were streaked on
petri-plates
[0103] in 10 mL of sterile distilled water a few cells were
dissolved by scrapping them from the plate with a loop,
[0104] Cells were sonicated for 541 and counted by Coulter Counter
(OD.sub.600 under 0.1),
[0105] About 4.times.10.sup.7 total cells were innuculated in 200
mL (giving 2-3.times.10.sup.5 cells/mL, an OD.sub.600 of about 0.6)
of YEPD medium (in an 1 liter flask),
[0106] Cells were grown for 16 h at 30.degree. C. under mild
shaking (about 6 generation) until about -1.times.10.sup.7 cells/mL
(OD.sub.600=2-3).
[0107] YEPD complete medium for routine growth of the cells before
transformation was made as follows: 1% yeast extract, 2% peptone,
2% glucose, 2% Bacto agar (for petri-plates) and distilled water.
All components were autoclaved for 20 min at 120.degree. C.
[0108] Transformation was performed as follows: A total of
2.times.10.sup.8 cells are used for a treatment (transformation of
the strain with a 1 mg of plasmid DNA), for example, a total of
1.times.10.sup.9 cells for 5 treatments (100 mL of culture broth
containing 1.times.10.sup.7 cells/ml). Transformation was carried
out through the lithium chloride method of Ito modified by Schiestl
and Gietz (R. H. Schiestl, R. D. Gietz,(1989), Current Genetics,
16:339-346). The transformants were plated on agar plates
(synthetic minimal medium) lacking leucine (to select for
plasmid-containing GRF18 cells) or lacking uracil (to select for
plasmid-containing X4004 cells) and grown at 30.degree. C. Single
colonies are stored either at 4.degree. C. by streaking them on
fresh selective plates or at -80.degree. C. in 15% glycerol.
[0109] All the transformations were performed in duplicate on the
basis of the following scheme:
2 Plasmid DNA (1 mL) yeast strain (2 .times. 10.sup.8 cells)
pEMBL-S0D374 X4004 pEMBLyex4 X4004 (negative control) pEMBL-SOD 374
GRF18 pEMBLyex4 GRF18 (negative control)
[0110] Strain GRF18 was grown on a synthetic medium without the
amino acid leucine for the selection of plasmid-bearing strains,
while in the case of X4004, the strain was grown on a synthetic
medium without uracile.
[0111] The induction of expression in both cases was performed by
shifting from minimal medium containing glucose to medium
containing galactose as the carbon source as follows. The
transformants, following streaking on plates, were grown in 50 mL
of synthetic minimal medium (lacking leucine for transformed GRF1 8
or uracil for transformed X4004) containing 2% of glucose, at
30.degree. C. for 12-14 h under mild shaking and aeration to reach
about 2.5-3.times.10.sup.7 cells/mL (OD.sub.6004-5). 50 mL of
selective synthetic medium, containing 2% of galactose, was
inoculated with 5.times.10.sup.7 cells (2 mL of preculture) to
reach about 1.times.10.sup.6 cells/mL. Growth was for about 17-18 h
(about 7 generations) at 30.degree. C. under mild shaking and
aeration to reach about 2-3.times.10.sup.7 cells/mL
(OD.sub.6004-5). 2.times.10.sup.8 cells (e.g. about 10 mL of
culture) were removed and used for protein extraction.
[0112] The medium to grow transformed cells was made as follows.
Synthetic selective medium (to grow transformed X4004 cells): 2% of
carbon source (glucose or galactose), 2% Bacto agar (for
petri-plates), 50 mg/liter of L-lysineHCl, 50 mg/liter of
L-methionine, and distilled water. Components were autoclaved for
20 min at 120.degree. C., difco yeast nitrogen base (YNB) without
amino acids was added (filtered concentrated stock solution
10.times. (67 g/liter)), 50 mg/mL of L-tryptophan (filtered
concentrated stock solution).
[0113] Synthetic selective medium (to grow transformed GRFL8
cells): 2% of carbon source (glucose or galactose), 2% Bacto agar
(for petri-plates), 50 mg/liter of L-histidine, distilled water.
Components were autoclave for 20 min at 120.degree. C., difco yeast
nitrogen base (YNB) without amino acids was added (filtered
concentrated stock solution 10.times. (67 g/liter)).
[0114] The ability of a microbial strain to produce a given
polypeptide may be tested through several approaches.
[0115] Initially, it is advisable to evaluate the production of the
simple polypeptide chain. In fact, the first question that has to
be answered is whether the expression machinery of the novel cell
is working efficiently in relation to the biosynthesis of the
desired chemical entity. Thus, to test the presence of large
amounts of a chemical entity like a polypeptide in cell extracts,
total protein extracts are first run on SDS denaturing
polyacrylamide gels which discriminate according to the molecular
weight of the polypeptide chain. In addition, since it can be
disposed of "isogenic" yeast strains (see negative controls), which
do not carry the SOD containing-expression plasmid, the
electrophoretic profile of a cell extract obtained from the
plasmid-bearing strain may be compared with the cell extract
obtained from a traditional strain.
[0116] Knowing the molecular weight of SOD (the SOD polypeptide
chain is composed of 154 amino acids with a corresponding molecular
weight of 15,700 Daltons), the two profiles were compared, in the
region of the gel corresponding to the molecular weight of SOD, for
the presence. Visualization was performed through non-specific
staining with Coomassie-blue.
[0117] Evaluation of the productivity of the clones was using the
analytical technique of SDS polyacrylamide electrophoresis as
follows.
[0118] a) Protein Yeast Extraction.
[0119] The protocol that was used for preparation of total protein
extracts from yeast, was partially modified from the method of
Jazwinski (S. Michail Jazwinski, 1990, Methods in Enzymology,
vol.182 p. 154) as follows:
[0120] 2.times.10.sup.8 cells were concentrated (in a 15 mL falcon
tube) by centrifugation (4000 rpm at 4.degree. C., 5 min),
[0121] The pellet was washed with sterile water at 4.degree. C.
(transferred to eppendorf vials),
[0122] The Pellet was resuspend in 400 microliters of 1.times. PBS
buffer,
[0123] 400 microliters of glass microbeads were added (prechilled
at -20.degree. C.),
[0124] 4 microliters (1 mg/mL) of pepstatin was added (protease
inibitor),
[0125] vortexed for 3 min (twice on ice),
[0126] 10 microliters of supernatant was stored at -20.degree. C.
for protein assay.
[0127] Staining of the gel with Coomassie blue gave a clear
electrophoretic band in the samples (GRF18 cells transformed with
plasmid pEMBL-SOD 374), corresponding to the molecular weight of
the Cu/Zn SOD yeast protein loaded on the same gel.
[0128] Such bands were not observed in the negative control samples
(GRF18 cells transformed with the plasmid pEMBLyex4 which does not
carry the SOD gene and X4004 cells transformed with the plasmid
pEMBLyex4 which does not carry the SOD gene). Approximately the
same quantity of protein extracts for each sample (about 1 mg) was
loaded on the polyacrylamide gel.
[0129] b) SOD Activity Determination by PIU-test.
[0130] The expression of a homologous or heterologous enzyme can be
determined by an appropriate activity test. Therefore, the
evaluation of the expression of yeast cells transformed by the two
molecular constructs was performed by quantification of the SOD
activity after induction of the cultures. The growth of the
cultures were monitored by determining cell numbers with a counter
(Coulter counter ZBI) (Lotti et al., Appl. Microbiol. Biotechnol.
28: 160-165 (1988)) or with measurement of absorbance at 600 nm.
The protein extracts were prepared as described under "protein
yeast extraction".
[0131] The presence of Cu/Zn SOD activity in total yeast cells
extracts was detected by the method of Marklund and Marklund, based
on the ability of the enzyme to inhibit the autoxidation of
pyrogallol (Marklund S. and Marklund G., Eur. J. Biochem.
47:469-474 (1974)).
[0132] The expression experiments were performed as batch
fermentation in complete synthetic medium.
[0133] Complete synthetic medium (Sherman F., Methods in
Enzymology. vol 194, Academic Press) with and without copper
(0.0025 g/liter) and zinc (0.05 g/liter) was prepared as follows
(in grams per liter): Bacto yeast Nitrogen base without amino acids
(Difco Laboratories, Detroit, Mich.), 6.7; carbon source (glucose
or galactose), 20; Adenine sulfate, 50; uracil, 50; L-tryptophan,
50; L-histidine, 50; L-arginine-HCl, 50; L-methionine, 50;
L-tyrosine, 50; L-isoleucine, 50; L-lysine-HCl, 50;
L-phenylalanine, 50; L-glutamic acid, 50; L-aspartic acid, 50;
L-valine, 50; L-threonine, 50; L-serine, 50.
[0134] Table 1 shows SOD activity data (in strain GRF18)of three
experiments and the mean values upon expression of the SOD gene
before deletion of MCS and bacterial sequences:
3TABLE I Transformants 1. PIU/mL 2. PIU/mL 3. PIU/mL Mean values
pEMBL SOD 374 575 168 180 301 (in GRF 18) pEMBLyex4 18 10 12 13 (in
GRF 18) Purchased Wine Yeast 22 -- 22
[0135] The average expression values are 301 PIU/mL for the vector
pEMBL-SOD 374, and 13 PIU/mL for the vector pEMBLyex4 (without the
insert). These values are important to judge the specific
expression of the target gene. They were determined in standardized
laboratory batch fermentations. The results show on average a 23
times elevated expression level compared to the original strain
(GRF 18 with plasmid but without insert). Compared to a purchased
wine yeast strain ("Seccoferm") a 22-fold increase in expression
was observed in the laboratory batch assays. It was shown that the
economy of the manufacturing process was highly increased by the
new constructions.
Example 6
[0136] Preparation of the Vector pEMBL-SOD 374 w/o MCS
[0137] The conclusion can be taken from Example 5 is that the
performance of the yeast strain GRF 18, containing the molecular
construct pEMBL-SOD 374, was very high, as control was the same
strain which did not contain any construct. Thus, the molecular
construct pEMBL-SOD 374 was used to build the final expression
system by deleting any non-yeast sequence.
[0138] The synthetic sequence of the "multiple cloning site" was
completely deleted by a digesting both ends of the polylinker with
the enzymes SstI and HindIII. This operation permitted the excision
of the entire 95 bases of the artificial sequence from the
molecular construct pEMBL-SOD 374 and allowed fpr insertion of the
complete yeast Cu/Zn SOD gene between the remaining natural
sites.
[0139] Detailed Protocol:
[0140] 1) The clone pEMBL-SOD 374 was cut by the enzymes HindIII
(New England Biolabs Inc., USA) and SacI (New England Biolabs Inc.,
USA) to excise the polylinker from the rest of the vector. The
enzyme HindIII recognizes the unique site "A/AGCTT" at one end of
the multiple cloning site, while the enzyme SacI is a isoschizomer
of the enzyme SstI and recognizes the same unique sequence
"GAGCT/C" at the other end of the multiple cloning site.
[0141] Following this double digestion, a HindIII-SacI fragment of
8705 bases which carries all the yeast and bacterial sequences of
the vector pEMBL without the polylinker, was isolated from agarose
gel by electroelution.
[0142] The 8705 bp-fragment was finally purified by
phenol-chloroform treatment and concentrated by ethanol
precipitation.
[0143] 2) The ends of the 8705 bp HindIII-SacI fragment were
treated with the enzyme Polymerase 1 "Klenow fragment" to create
compatible ends on the fragment for further manipulations. This
treatment creates blunts ends on the fragment by filling of the
HindIII end (which is the 5' protuding termini) and cutting of the
SacI end (which is the 3' protuding termini).
[0144] Finally, the blunt-ended fragment was purified by
phenol-chloroform and ethanol precipitation.
[0145] 3) The DNA fragment carrying the yeast Cu/Zn SOD gene
present on pEMBL-SOD 374 was cut with the enzymes EcoRI (New
England Biolabs Inc., USA) and SalI (New England Biolabs Inc., USA)
which recognize respectively the sites "G/AATTC" and "G/TCGAC".
This double digestion permits the isolation of a fragment of 916 bp
which carries a 342 bp yeast upstream sequence, the entire open
reading frame of the yeast Cu/Zn SOD gene of 462 bp and a yeast
downstream sequence of 112 bp. This 916 bp fragment, which does not
carry any other bacterial or artificial sequence, was isolated from
agarose gel by electroelution. The fragment was further purified by
phenol-chloroform treatment and ethanol precipitation.
[0146] 4) After purification, the EcoRI-SalI fragment was treated
with the enzyme Polymerase I "Klenow fragment" which performs the
filling of the EcoRI and SalI ends (which are both 5' protuding
termini) to make them compatible to the subcloned ends of the
purified pEMBL vector from which the polylinker was previously
deleted. The blunt-ended fragment was again purified by
phenol-chloroform and ethanol precipitation.
[0147] 5) The blunt-ended 916 bp fragment containing the yeast
Cu/Zn SOD gene was finally subcloned into the blunt-ended 8705 bp
fragment-pEMBL vector by ligation with the enzyme T4 DNA Ligase
(Boehringer Mannheim GmbH, Mannheim). The ligation was performed at
16.degree. C. for 20 h.
[0148] 6) After transformation of the ligation mixture (blunt-ended
916 bp fragment plus blunt-ended 8705 bp fragment) in E. coli (XL
1-Blue strain), a random screen was performed to isolate the clones
which carry the right sequences. A number of bacterial clones from
the transformation were grown overnight at 37.degree. C. in LB
broth medium and the DNA plasmids from the clones was extracted by
the "mini prep" method ("Wizard" minipreps DNA purification system,
Promega Corp., USA).
[0149] PCR amplification as well as DNA sequencing were used to
verify both the presence and the correct orientation of the
fragment containing the yeast Cu/Zn SOD gene in the clones.
[0150] The correctness was confirmed by sequencing and agarose gel
electrophoresis.
Example 7
[0151] Preparation of the Final Vector pEMBL-SOD w/o MCS w/o
Coli
[0152] The clone "pEMBL-SOD 374 w/o MCS" obtained from Example 6
carries only the yeast sequences (yeast funtional sequences and
yeast Cu/Zn SOD gene) and the bacterial sequences. The non-yeast
part of the "pEMBL-SOD 374 w/o MCS" clone carries the origin of
replication for the bacterium Escherichia coli, the ampicilline
bacterial selectable marker and the origin of replication derived
from the filamentous bacteriophage f1.
[0153] This region covers more than 4000 bases and must be deleted
by enzymatic digestion in order to obtain a final vector which
carries only yeast sequences. The final vector following such a
manipulation will be able to replicate only in yeast strains. In
fact, the only sequences present in the final vector are:
[0154] the Leu 2-d yeast selectable marker useful to select the
yeast cell transformants,
[0155] the origin of replication of 2-micron yeast episome that
provides high mitotic stability and replication of the plasmid,
[0156] the entire yeast expression hybrid UAS GAL/CYC system that
provides high level of transcription of the gene under its
control,
[0157] the yeast SOD gene (with upstream and downstream functional
yeast sequences) which produces the enzyme Cu/Zn Superoxide
Dismutase.
[0158] The following is a detailed protocol for preparation of
vector.
[0159] 1) The clone "pEMBL-SOD 374 w/o MCS", previously deleted
from the multiple cloning site, was digested with the blunt
endonucleases StuI (in position 5942 of the original pEMBLyex4
vector) and NruI (in position 1791 of the original pEMBLyex4
vector). The enzyme StuI (Pharmacia, Uppsala) recognizes the unique
blunt site "AGG/CCT" and the enzyme NruI (Pharmacia, Uppsala)
recognizes the unique blunt site "TCG/CGA". The resulting StuI-NruI
fragment of 4689 bp carrying all the bacterial sequences was
separated from the rest of the pEMBL vector by gel electrophoresis.
The double digestion also deletes part of the yeast selectable
marker URA 3 on the vector but it preserves the complete function
of the other yeast selectable marker LEU-2d, which may be used for
the selection of clones during further manipulations.
[0160] 2) The fragment pEMBL vector containing only yeast sequences
and the yeast Cu/Zn SOD gene were isolated from agarose gel by
electroelution and purified by phenol-chloroform and ethanol
precipitation.
[0161] 3) The ends of the purified pEMBL vector (both ends are
blunt) were rejoined together by the enzyme T4 DNA ligase
(Boehringer Mannheim GmbH, Mannheim) and the ligation mixture was
used to transform the yeast strain GRF 18.
[0162] 4) Clones from the transformation were plated on agar plates
with synthetic minimal medium lacking leucine for selection (by the
selectable marker Leu 2-d) of the GRF 18 cells containing the
plasmid.
[0163] 5) A number of yeast colonies obtained from the
transformation were tested by PCR to confirm both the presence of
the yeast fragment containing the yeast Cu/Zn SOD gene and the
absence of any bacterial sequence between the StuI and NruI
enzymatic sites. To confirm the presence of the yeast fragment on
the final construct "pEMBL-SOD w/o MCS w/o Coli", PCR amplification
was performed with the two flanking primers SOD proA and SOD proB.
An electrophoretic band of about 1133 bp length confirmed the
presence of the fragment containing the yeast Cu/Zn SOD gene in six
yeast clones.
[0164] The correctness was confirmed by sequencing and agarose gel
electrophoresis.
[0165] The final vector "pEMBL-SOD w/o MCS w/o Coli" derived from
such manipulations does not carry any bacterial or artificial
sequence and will be able to replicate only in yeast strains
because of the presence of only yeast sequences. The final vector
is presented as a restriction map in FIG. 3.
Example 8
[0166] Evaluation of the Expression of the Yeast Cu/Zn SOD Gene in
the New Yeast Strain
[0167] This example relates to the evaluation of the expression of
the yeast Cu/Zn SOD gene in the new yeast strains as constructed in
Examples 6 and 7. All the experiments were performed as in Example
5 by growing yeast cells in complete synthetic media.
[0168] Table 2 shows the presence of SOD activity in total yeast
cell extracts upon expression of SOD gene after deletion of MCS and
bacterial sequences in GRF 18-pEMBL SOD 374 in complete synthetic
medium.
4 TABLE 2 Transformants PIU/mL pEMBL-SOD 374 (in GRE 18) 138
pEMBLyex4 (in GRE 18) 17 pEMBL-SODw/oMCSw/oColi (in GRE 18) 141
[0169] The test results indicated that the vector
pEMBL-SODw/oMCSw/oColi expresses the yeast Cu/Zn SOD gene in higher
yield compared to vector pEMBLyex4 (in GRF 18).
Example 9
[0170] Isolation and Purification of Yeast Cu/Zn SOD
[0171] The production of the target polypeptide, such as the yeast
superoxide dismutase, is performed under aerobic conditions in
computer-controlled fermenters, for example, according to
Alberghina et al. ibid. Controlled parameters of fermentation are
temperature, dissoluted oxygen, pH and ethanol concentration.
[0172] The purification of the polypeptide is comprised the
following steps:
[0173] 1. The yeast cells in the fermentation broth is directly
lysed by homogenization with a homogenizer (e. g. an APV Gaulin) at
a temperature of between 20 and 30.degree. C., a pressure of
between 600 and 800 bar and 3 cyles, or with a dynobed mill (e. g.
a Dyno Mill Model KDL) filled with acid washed 0.3 mm diameter, and
recirculating the suspension through the mill at 160 mI/mm for 1-2
mm at room temperature.
[0174] 2. The separation of cell debris and proteins is performed
by centrifugation (e. g. with a Beckmann J2-21 centrifuge with
JA-10 fixed angle rotor for 60 mm at a speed of 14,000 g), or by
microfiltration, (e. g. by tangential flow filtration with Minitan
(Millipore Corp., Bedford)) equipped with a 0.45 pm cut-off
membrane.
[0175] 3. Protein was concentrated and buffer exchanged by
ultrafiltration and diafiltration (for example Tangential flow
filtration with Minitan (Millipore Corp., Bedford)) equipped with a
10,000 Dalton cut-off cellulose membrane (PLGCOMP 04 membrane).
Exchange Buffer was 20 mM Tris-HCl pH 8.0.
[0176] 4. Purification by cationic exchange chromatography (e. g.
with DEAE-Sepharose). Loading buffer: 20 mM Tris-HCl pH 8.0.
Elution buffer: 20 mM Tris-HCl pH 8.0, 1 M NaCl.
[0177] 5. Concentration and buffer change by ultrafiltration and
diafiltration to obtain the final buffered SOD solution.
Conditions: a Tangential flow filtration with Minitan (Millipore
Corp., Bedford) equipped with a 10,000 Dalton cut-off membrane.
Buffer: 20 mM Tris-HCl, pH 8.0.
[0178] At the end of fermentation the activity is at least 5000
PIU/mL. The yield obtainable with the new strain according to the
invention is at least 10 times higher compared to the yield
obtainable with purchasable bakers yeast. From 1 g bakers yeast
5000 PIU can be isolated, whereas at least 40,000 PIU can be
isolated from 1 g pEMBL-SOD 374 GRF 18 yeast.
Sequence CWU 1
1
3 1 22 DNA Artificial Sequence Oligonucleotide Primer 1 ggacgtaagc
atctctgaag tg 22 2 28 DNA Artificial Sequence Oligonucleotide
Primer 2 gccgtcgacg gacccctcaa gacccctc 28 3 30 DNA Artificial
Sequence Oligonucleotide Primer 3 gccgtcgaca cacttggtga atgatcaagg
30
* * * * *