 |
Description  |
|
|
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a purified thermostable enzyme. In one
embodiment the enzyme is DNA polymerase purified from Thermus aquaticus
and has a molecular weight of about 86,000-90,000.
2. Background Art
Extensive research has been conducted on the isolation of DNA polymerases
from mesophilic microorganisms such as E. coli. See, for example, Bessman
et al., J. Biol. Chem. (1957) 233:171-177 and Buttin and Kornberg (1966)
J. Biol. Chem. 241:5419-5427.
In contrast, relatively little investigation has been made on the isolation
and purification of DNA polymerases from thermophiles, such as Thermus
aquaticus. Kaledin et al., Biokhymiya (1980) 45:644-651 discloses a
six-step isolation and purification procedure of DNA polymerase from cells
of T. aquaticus YT1 strain. These steps involve isolation of crude
extract, DEAE-cellulose chromatography, fractionation on hydroxyapatite,
fractionation on DEAE-cellulose, and chromatography on single-strand
DNA-cellulose. The pools from each stage were not screened for
contaminating endo- and exonuclease(s). The molecular weight of the
purified enzyme is reported as 62,000 daltons per monomeric unit.
A second purification scheme for a polymerase from T. aquaticus is
described by A. Chien et al., J. Bacteriol. (1976) 127:1550-1557. In this
process, the crude extract is applied to a DEAE-Sephadex column. The
dialyzed pooled fractions are then subjected to treatment on a
phosphocellulose column. The pooled fractions are dialyzed and bovine
serum albumin (BSA) is added to prevent loss of polymerase activity. The
resulting mixture is loaded on a DNA-cellulose column. The pooled material
from the column is dialyzed and analyzed by gel filtration to have a
molecular weight of about 63,000 daltons, and, by sucrose gradient
centrifugation of about 68,000 daltons.
The use of a thermostable enzyme to amplify existing nucleic acid sequences
in amounts that are large compared to the amount initially present has
been suggested in copending U.S. Pat. No. 4,683,195. Primers, nucleotide
triphosphates, and a polymerase are used in the process, which involves
denaturation, synthesis of template strands and hybridization. The
extension product of each primer becomes a template for the production of
the desired nucleic acid sequence. The application discloses that if the
polymerase employed is a thermostable enzyme, it need not be added after
every denaturation step, because the heat will not destroy its activity.
No other advantages or details are provided on the use of a purified
thermostable DNA polymerase. Furthermore, New England Biolabs had marketed
a polymerase from T. aquaticus, but discovered that the polymerase
activity decreased substantially with time in a storage buffer not
containing non-ionic detergents.
Accordingly, there is a desire in the art to produce a purified, stable
thermostable enzyme that may be used to improve the diagnostic
amplification process described above.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a purified thermostable enzyme
that catalyzes combination of nucleotide triphosphates to form a nucleic
acid strand complementary to a nucleic acid template strand. Preferably
the purified enzyme is DNA polymerase from Thermus aquaticus and has a
molecular weight of about 86,000-90,000 daltons. This purified material
may be used in a temperature-cycling amplification reaction wherein
nucleic acid sequences are produced from a given nucleic acid sequence in
amounts that are large compared to the amount initially present so that
they can be detected easily.
The gene encoding the enzyme from DNA polymerase from Thermus aquaticus has
also been identified and provides yet another means to retrieve the
thermostable enzyme of the present invention. In addition to the gene
encoding the approximately 86,000-90,000 dalton enzyme, gene derivatives
encoding DNA polymerase activity are also presented.
Finally, the invention also encompasses a stable enzyme composition
comprising a purified, thermostable enzyme as described above in a buffer
containing one or more non-ionic polymeric detergents.
The purified enzyme, as well as the enzymes produced by recombinant DNA
techniques, provide much more specificity than the Klenow fragment, which
is not thermostable. In addition, the purified enzyme and the
recombinantly produced enzymes exhibit the appropriate activity expected
when dTTP or other nucleotide triphosphates are not present in the
incubation mixture with the DNA template. Also, the enzymes herein have a
broader pH profile than that of the thermostable enzyme from Thermus
aquaticus described in the literature, with more than 50% of the activity
at pH 7 as at pH 8. Finally, the thermostable enzyme herein can be stored
in a buffer with non-ionic detergents so that it is stable, not losing
activity over a period of time.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a restriction site map of plasmid pFC83 that contains the
.about.4.5 kb HindIII T. aquaticus DNA insert subcloned into plasmid
BSM13+.
FIG. 2 is a restriction site map of plasmid pFC85 that contains the
.about.2.8 kb HindIII to Asp718 T. aquaticus DNA insert subcloned into
plasmid BSM13+.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As used herein, "cell", "cell line", and "cell culture" can be used
interchangeably and all such designations include progeny. Thus, the words
"transformants" or "transformed cells" includes the primary subject cell
and cultures derived therefrom without regard for the number of transfers.
It is also understood that all progeny may not be precisely identical in
DNA content, due to deliberate or inadvertent mutations. Mutant progeny
that have the same functionality as screened for in the originally
transformed cell are included.
The term "control sequences" refers to DNA sequences necessary for the
expression of an operably linked coding sequence in a particular host
organism. The control sequences that are suitable for procaryotes, for
example, include a promoter, optionally an operator sequence, a ribosome
binding site, and possibly, other as yet poorly understood sequences.
Eucaryotic cells are known to utilize promoters, polyadenylation signals,
and enhancers.
The term "expression system" refers to DNA sequences containing a desired
coding sequence and control sequences in operable linkage, so that hosts
transformed with these sequences are capable of producing the encoded
proteins. In order to effect transformation, the expression system may be
included on a vector; however, the relevant DNA may then also be
integrated into the host chromosome.
The term "gene" as used herein refers to a DNA sequence that encodes a
recoverable bioactive polypeptide or precursor. The polypeptide can be
encoded by a full-length gene sequence or any portion of the coding
sequence so long as the enzymatic activity is retained.
"Operably linked" refers to juxtaposition such that the normal function of
the components can be performed. Thus, a coding sequence "operably linked"
to control sequences refers to a configuration wherein the coding
sequences can be expressed under the control of the sequences.
"Non-ionic polymeric detergents" refers to surface-active agents that have
no ionic charge and that are characterized, for purposes of this
invention, by their ability to stabilize the enzyme herein at a pH range
of from about 3.5 to about 9.5, preferably from 4 to 8.5.
The term "oligonucleotide" as used herein is defined as a molecule
comprised of two or more deoxyribonucleotides or ribonucleotides,
preferably more than three. Its exact size will depend on many factors,
which in turn depend on the ultimate function or use of the
oligonucleotide. The oligonucleotide may be derived synthetically or by
cloning.
The term "primer" as used herein refers to an oligonucleotide, whether
occurring naturally as in a purified restriction digest or produced
synthetically, which is capable of acting as a point of initiation of
synthesis when placed under conditions in which synthesis of a primer
extension product which is complementary to a nucleic acid strand is
induced, i.e., in the presence of four different nucleotide triphosphates
and thermostable enzyme in an appropriate buffer ("buffer" includes pH,
ionic strength, cofactors, etc.) and at a suitable temperature. For Taq
polymerase the buffer herein preferably contains 1.5-2 mM of a magnesium
salt, preferably MgCl.sub.2, 150-200 .mu.M of each nucleotide, and 1 .mu.M
of each primer, along with preferably 50 mM KCl, 10 mM Tris buffer, pH
8-8.4, and 100 .mu.g/ml gelatin.
The primer is preferably single-stranded for maximum efficiency in
amplification, but may alternatively be double-stranded. If
double-stranded, the primer is first treated to separate its strands
before being used to prepare extension products. Preferably, the primer is
an oligodeoxyribonucleotide. The primer must be sufficiently long to prime
the synthesis of extension products in the presence of the thermostable
enzyme. The exact lengths of the primers will depend on many factors,
including temperature, source of primer and use of the method. For
example, depending on the complexity of the target sequence, the
oligonucleotide primer typically contains 15-25 nucleotides, although it
may contain more or fewer nucleotides. Short primer molecules generally
require cooler temperatures to form sufficiently stable hybrid complexes
with template.
The primers herein are selected to be "substantially" complementary to the
different strands of each specific sequence to be amplified. This means
that the primers must be sufficiently complementary to hybridize with
their respective strands. Therefore, the primer sequence need not reflect
the exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer, with the
remainder of the primer sequence being complementary to the strand.
Alternatively, non-complementary bases or longer sequences can be
interspersed into the primer, provided that the primer sequence has
sufficient complementarity with the sequence of the strand to be amplified
to hybridize therewith and thereby form a template for synthesis of the
extension product of the other primer. However, for detection purposes,
particularly using labeled sequence-specific probes, the primers typically
have exact complementarity to obtain the best results.
As used herein, the terms "restriction endonucleases" and "restriction
enzymes" refer to bacterial enzymes each of which cut double-stranded DNA
at or near a specific nucleotide sequence.
As used herein, the term "thermostable enzyme" refers to an enzyme which is
stable to heat and is heat resistant and catalyzes (facilitates)
combination of the nucleotides in the proper manner to form the primer
extension products that are complementary to each nucleic acid strand.
Generally, the synthesis will be initiated at the 3' end of each primer
and will proceed in the 5' direction along the template strand, until
synthesis terminates, producing molecules of different lengths. There may
be a thermostable enzyme, however, which initiates synthesis at the 5' end
and proceeds in the other direction, using the same process as described
above.
The thermostable enzyme herein must satisfy a single criterion to be
effective for the amplication reaction, i.e., the enzyme must not become
irreversibly denatured (inactivated) when subjected to the elevated
temperatures for the time necessary to effect denaturation of
double-stranded nucleic acids. Irreversible denaturation for purposes
herein refers to permanent and complete loss of enzymatic activity. The
heating conditions necessary for denaturation will depend, e.g., on the
buffer salt concentration and the length and nucleotide composition of the
nucleic acids being denatured, but typically range from about 90 to about
105.degree. C. for a time depending mainly on the temperature and the
nucleic acid length, typically about 0.5 to four minutes. Higher
temperatures may be tolerated as the buffer salt concentration and/or GC
composition of the nucleic acid is increased. Preferably, the enzyme will
not become irreversibly denatured at about 90-100.degree. C.
The thermostable enzyme herein preferably has an optimum temperature at
which it functions that is higher than about 40.degree. C., which is the
temperature below which hybridization of primer to template is promoted,
although, depending on (1) magnesium and salt concentrations and (2)
composition and length of primer, hybridization can occur at higher
temperature (e.g., 45-70.degree. C.). The higher the temperature optimum
for the enzyme, the greater the specificity and/or selectivity of the
primer-directed extension process. However, enzymes that are active below
40.degree. C., e.g., at 37.degree. C., are also within the scope of this
invention provided they are heat-stable. Preferably, the optimum
temperature ranges from about 50.degree. to 90.degree. C., more preferably
60-80.degree. C.
The thermostable enzyme herein may be obtained from any source and may be a
native or recombinant protein. Examples of enzymes that have been reported
in the literature as being resistant to heat include heat-stable
polymerases, such as, e.g., polymerases extracted from the thermophilic
bacteria Thermus flavus, Thermus ruber, Thermus thnrmophilus, Bacillus
stearothermophilus (which has a somewhat lower temperature optimum than
the others listed), Thermus aquaticus, Thermus lacteus, Thermus rubens,
and Methanothermus fervidus.
The preferred thermostable enzyme herein is a DNA polymerase isolated from
Thermus aquaticus. Various strains thereof are available from the American
Type Culture Collection, Rockville, Md., and are described by T. D. Brock,
J. Bact. (1969) 98:289-297, and by T. Oshima, Arch. Microbiol. (1978) 117:
189-196. One of these preferred strains is strain YT-1.
For recovering the native protein the cells are grown using any suitable
technique. One such technique is described by Kaledin et al., Biokhimiya
(1980), supra, the disclosure of which is incorporated herein by
reference. Briefly, the cells are grown on a medium, in one liter, of
nitrilotriacetic acid (100 mg), tryptone (3 g), yeast extract (3 g),
succinic acid (5 g), sodium sulfite (50 mg), riboflavin (1 mg), K.sub.2
HPO.sub.4 (522 mg), MgSO.sub.4 (480 mg), CaCl.sub.2 (222 mg), NaCl (20
mg), and trace elements. The pH of the medium is adjusted to 8.0.+-.0.2
with KOH. The yield is increased if cultivated with vigorous aeration up
to 20 g/liter of cells at a temperature of 70.degree. C. Cells in the late
logarithmic stage (determined by absorbance at 550 nm) are collected by
centrifugation, washed with a buffer and stored frozen at -20.degree. C.
In another method for growing the cells, described in Chien et al., J.
Bacteriol. (1976), supra, the disclosure of which is incorporated herein
by reference, a defined mineral salts medium containing 0.3% glutamic acid
supplemented with 0.1 mg/1 biotin, 0.1 mg/1 thiamine, and 0.05 mg/l
nicotinic acid is employed. The salts include nitrilotriacetic acid,
CaSO.sub.4, MgSO.sub.4, NaCl, KNO.sub.3, NaNO.sub.3, ZnSO.sub.4, H.sub.3
BO.sub.3, CuSO.sub.4, NaMoO.sub.4, CoCl.sub.2, FeCl.sub.3, MnSO.sub.4, and
Na.sub.2 HPO.sub.4. The pH of the medium is adjusted to 8.0 with NaOH.
In the Chien et al. technique, the cells are grown initially at 75.degree.
C. in a water bath shaker. On reaching a certain density, 1 liter of these
cells is transferred to 16-liter carbons which are placed in hot-air
incubators. Sterile air is bubbled through the cultures and the
temperature maintained at 75.degree. C. The cells are allowed to grow for
20 hours before being collected by centrifugation.
After cell growth, the isolation and purification of the enzyme take place
in six stages, each of which is carried out at a temperature below room
temperature, preferably about 4.degree. C.
In the first stage or step, the cells, if frozen, are thawed, disintegrated
by ultrasound, suspended in a buffer at about pH 7.5, and centrifuged.
In the second stage, the supernatant is collected and then fractionated by
adding a salt such as dry ammonium sulfate. The appropriate fraction
(typically 45-75% of saturation) is collected, dissolved in a 0.2M
potassium phosphate buffer preferably at pH 6.5, and dialyzed against the
same buffer.
The third step removes nucleic acids and some protein. The fraction from
the second stage is applied to a DEAE-cellulose column equilibrated with
the same buffer as used above. Then the column is washed with the same
buffer and the flow-through protein-containing fractions, determined by
absorbance at 280 nm, are collected and dialyzed against a 10 mM potassium
phosphate buffer, preferably with the same ingredients as the first
buffer, but at a pH of 7.5.
In the fourth step, the fraction so collected is applied to a
hydroxyapatite column equilibrated with the buffer used for dialysis in
the third step. The column is then washed and the enzyme eluted with a
linear gradient of a buffer such as 0.01M to 0.5M potassium phosphate
buffer at pH 7.5 containing 10 mM 2-mercaptoethanol and 5% glycerine. The
pooled fractions containing thermostable enzyme (e.g., DNA polymerase)
activity are dialyzed against the same buffer used for dialysis in the
third step.
In the fifth stage, the dialyzed fraction is applied to a DEAE-cellulose
column, equilibrated with the buffer used for dialysis in the third step.
The column is then washed and the enzyme eluted with a linear gradient of
a buffer such as 0.01 to 0.6M KCl in the buffer used for dialysis in the
third step. Fractions with thermostable enzyme activity are then tested
for contaminating deoxyribonucleases (endo- and exonucleasesl using any
suitable procedure. For example, the endonuclease activity may be
determined electrophoretically from the change in molecular weight of
phage .lambda. DNA or supercoiled plasmid DNA after incubation with an
excess of DNA polymerase. Similarly, exonuclease activity may be
determined electrophoretically from the change in molecular weight of DNA
after treatment with a restriction enzyme that cleaves at several sites.
The fractions determined to have no deoxyribonuclease activity are pooled
and dialyzed against the same buffer used in the third step.
In the sixth step, the pooled fractions are placed on a phosphocellulose
column with a set bed volume. The column is washed and the enzyme eluted
with a linear gradient of a buffer such as 0.01 to 0.4M KCl in a potassium
phosphate buffer at pH 7.5. The pooled fractions having thermostable
polymerase activity and no deoxyribonuclease activity are dialyzed against
a buffer at pH 8.0.
The molecular weight of the dialyzed product may be determined by any
technique, for example, by SDS PAGE using protein molecular weight
markers. The molecular weight of one of the preferred enzymes herein, the
DNA polymerase purified from Thermus aquaticus, is determined by the above
method to be about 86,000-90,000 daltons.
The thermostable enzyme of this invention may also be produced by
recombinant DNA techniques, as the gene encoding this enzyme has been
cloned from Thermus aquaticus genomic DNA. The complete coding sequence
for the Thermus aquaticus (Taq) polymerase can be derived from
bacteriophage CH35:Taq#4-2 on an approximately 3.5 kilobase (kb)
BglII-Asp718 (partial) restriction fragment contained within an .about.18
kb genomic DNA insert fragment. This bacteriophage was deposited with the
American Type Culture Collection (ATCC) on May 28, 1987 and has accession
No. ATCC 40336. Alternatively, the gene can be constructed by ligating an
.about.750 base pair (bp) BglII-HindIII restriction fragment isolated from
plasmid pFC83 (ATCC 67422 deposited May 28, 1987) to an .about.2.8 kb
HindIII-Asp718 restriction fragment isolated from plasmid pFC85 (ATCC
67421 deposited May 28, 1987). The pFC83 restriction fragment comprises
the amino-terminus encoding region of the Taq polymerase gene while the
restriction fragment from pFC85 comprises the carboxyl-terminus encoding
region. Thus, ligation of these two fragments into a correspondingly
digested vector with appropriate control sequences will result in the
translation of a full-length Taq polymerase.
It has also been found that the entire coding sequence of the Taq
polymerase gene is not required to recover a biologically active gene
product with the desired enzymatic activity. Amino-terminal deletions
wherein approximately one-third of the coding sequence is absent have
resulted in producing a gene product that is quite active in polymerase
assays.
In addition to the N-terminal deletions, individual amino acid residues in
the peptide chain comprising Taq polymerase may be modified by oxidation,
reduction, or other derivatization, and the protein may be cleaved to
obtain fragments that retain activity. Such alterations that do not
destroy activity do not remove the DNA sequence encoding such protein from
the definition of gene.
Thus, modifications to the primary structure itself by deletion, addition,
or alteration of the amino acids incorporated into the sequence during
translation can be made without destroying the activity of the protein.
Such substitutions or other alterations result in proteins having an amino
acid sequence encoded by DNA falling within the contemplated scope of the
present invention.
Polyclonal antiserum from rabbits immunized with the purified 86,000-90,000
dalton polymerase of this invention was used to probe a Thermus aquaticus
partial genomic expression library to obtain the appropriate coding
sequence as described below. The cloned genomic sequence can be expressed
as a fusion polypeptide, expressed directly using its own control
sequences, or expressed by constructions using control sequences
appropriate to the particular host used for expression of the enzyme.
Of course, the availability of DNA encoding these sequences provides the
opportunity to modify the codon sequence so as to generate mutein forms
also having DNA polymerase activity.
Thus, these tools can provide the complete coding sequence for Taq DNA
polymerase from which expression vectors applicable to a variety of host
systems can be constructed and the coding sequence expressed. It is also
evident from the foregoing that portions of the Taq polymerase-encoding
sequence are useful as probes to retrieve other thermostable
polymerase-encoding sequences in a variety of species. Accordingly,
portions of the genomic DNA encoding at least six amino acids can be
replicated in E. coli and the denatured forms used as probes or
oligodeoxyribonucleotide probes can be synthesized which encode at least
six amino acids and used to retrieve additional DNAs encoding a
thermostable polymerase. Because there may not be a precisely exact match
between the nucleotide sequence in the Thermus aquaticus form and that in
the corresponding portion of other species, oligomers containing
approximately 18 nucleotides (encoding the six amino acid stretch) are
probably necessary to obtain hybridization under conditions of sufficient
stringency to eliminate false positives. The sequences encoding six amino
acids would supply information sufficient for such probes.
Suitable Hosts, Control Systems and Methods
In general terms, the production of a recombinant form of Taq polymerase
typically involves the following:
First, a DNA is obtained that encodes the mature (used here to include all
muteins) enzyme or a fusion of the Taq polymerase to an additional
sequence that does not destroy its activity or to an additional sequence
cleavable under controlled conditions (such as treatment with peptidase)
to give an active protein. If the sequence is uninterrupted by introns it
is suitable for expression in any host. This sequence should be in an
excisable and recoverable form.
The excised or recovered coding sequence is then preferably placed in
operable linkage with suitable control sequences in a replicable
expression vector. The vector is used to transform a suitable host and the
transformed host cultured under favorable conditions to effect the
production of the recombinant Taq polymerase. Optionally the Taq
polymerase is isolated from the medium or from the cells; recovery and
purification of the protein may not be necessary in some instances, where
some impurities may be tolerated.
Each of the foregoing steps can be done in a variety of ways. For example,
the desired coding sequences may be obtained from genomic fragments and
used directly in appropriate hosts. The constructions for expression
vectors operable in a variety of hosts are made using appropriate
replicons and control sequences, as set forth below. Suitable restriction
sites can, if not normally available, be added to the ends of the coding
sequence so as to provide an excisable gene to insert into these vectors.
The control sequences, expression vectors, and transformation methods are
dependent on the type of host cell used to express the gene. Generally,
procaryotic, yeast, insect or mammalian cells are presently useful as
hosts. Procaryotic hosts are in general the most efficient and convenient
for the production of recombinant proteins and therefore preferred for the
expression of Taq polymerase.
In the particular case of Taq polymerase, evidence indicates that
considerable deletion at the N-terminus of the protein may occur under
both recombinant and native conditions, and that the activity of the
protein is still retained. It appears that the native proteins isolated
may be the result of proteolytic degradation, and not translation of a
truncated gene. The mutein produced from the truncated gene of plasmid
pFC85 is, however, fully active in assays for DNA polymerase, as is that
produced from DNA encoding the full-length sequence. Since it is clear
that certain N-terminal shortened forms are active, the gene constructs
used for expression of the polymerase may also include the corresponding
shortened forms of the coding sequence.
Control Sequences and Corresponding Hosts
Procaryotes most frequently are represented by various strains of E. coli.
However, other microbial strains may also be used, such as bacilli, for
example, Bacillus subtilis, various species of Pseudomonas, or other
bacterial strains. In such procaryotic systems, plasmid vectors that
contain replication sites and control sequences derived from a species
compatible with the host are used. For example, E. coli is typically
transformed using derivatives of pBR322, a plasmid derived from an E. coli
species by. Bolivar, et al., Gene (1977) 2:95. pBR322 contains genes for
ampicillin and tetracycline resistance, and thus provides additional
markers that can be either retained or destroyed in constructing the
desired vector. Commonly used procaryotic control sequences, which are
defined herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site sequences,
include such commonly used promoters as the .beta.-lactamase
(penicillinase) and lactose (lac) promoter systems (Chang, et al., Nature
(1977) 198:1056), the tryptophan (trp) promoter system (Goeddel, et al.,
Nucleic Acids Res. (1980) 8:4057) and the lambda-derived P.sub.L promoter
(Shimatake, et al., Nature (1981) 292:128) and N-gene ribosome binding
site, which has been made useful as a portable control cassette (as set
forth in U.S. Pat. No. 4,711,845, filed Dec. 24, 1984), which comprises a
first DNA sequence that is the P.sub.L promoter operably linked to a
second DNA sequence corresponding to N.sub.RBS upstream of a third DNA
sequence having at least one restriction site that permits cleavage within
six bp 3' of the N.sub.RBS sequence. Also useful is the phosphatase A
(phoA) system described by Chang, et al. in European Patent Publication
No. 196,864 published Oct. 8, 1986, assigned to the same assignee and
incorporated herein by reference. However, any available promoter system
compatible with procaryotes can be used.
In addition to bacteria, eucaryotic microbes, such as yeast, may also be
used as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker's
yeast, are most used, although a number of other strains are commonly
available. While vectors employing the 2 micron origin of replication are
illustrated (Broach, J. R., Meth. Enz. (1983) 101:307), other plasmid
vectors suitable for yeast expression are known (see, for example,
Stinchcomb, et al., Nature (1979) 282:39, Tschempe, et al., Gene (1980)
10:157 and Clarke, L., et al., Meth. Enz. (1983) 101:300). Control
sequences for yeast vectors include promoters for the synthesis of
glycolytic enzymes (Hess, et al., J. Adv. Enzyme Reg. (1968) 7:149;
Holland, et al., Biotechnology (1978) 17:4900).
Additional promoters known in the art include the promoter for
3-phosphoglycerate kinase (Hitzeman, et al., J. Biol. Chem. (1980)
255.2073), and those for other glycolytic enzymes, such as
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
phosphoglucose isomerase, and glucokinase. Other promoters that have the
additional advantage of transcription controlled by growth conditions are
the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid
phosphatase, degradative enzymes associated with nitrogen metabolism, and
enzymes responsible for maltose and galactose ultilization (Holland,
supra).
It is also believed that terminator sequences are desirable at the 3' end
of the coding sequences. Such terminators are found in the 3' untranslated
region following the coding sequences in yeast-derived genes. Many of the
vectors illustrated contain control sequences derived from the enolase
gene containing plasmid peno46 (Holland, M. J., et al., J. Biol. Chem.
(1981) 256:1385) or the LEU2 gene obtained from YEp13 (Broach, J., et al.,
Gene (1978) 8:121); however, any vector containing a yeast-compatible
promoter, origin of replication, and other control sequences is suitable.
It is also, of course, possible to express genes encoding polypeptides in
eucaryotic host cell cultures derived from multicellular organisms. See,
for example, Tissue Culture, Academic Press, Cruz and Patterson, editors
(1973). Useful host cell lines include murine myelomas N51, VERO and HeLa
cells, and Chinese hamster ovary (CHO) cells. Expression vectors for such
cells ordinarily include promoters and control sequences compatible with
mammalian cells such as, for example, the commonly used early and late
promoters from Simian Virus 40 (SV 40) (Fiers, et al., Nature (1978)
273:113), or other viral promoters such as those derived from polyoma,
Adenovirus 2, bovine papiloma virus, or avian sarcoma viruses, or
immunoglobulin promoters and heat shock promoters. A system for expressing
DNA in mammalian systems using the BPV as a vector is disclosed in U.S.
Pat. No. 4,419,446. A modification of this system is described in U.S.
Pat. No. 4,601,978. General aspects of mammalian cell host system
transformations have been described by Axel, U.S. Pat. No. 4,399,216. It
now appears, also, that "enhancer" regions are important in optimizing
expression; these are, generally, sequences found upstream of the promoter
region. Origins of replication may be obtained, if needed, from viral
sources. However, integration into the chromosome is a common mechanism
for DNA replication in eucaryotes.
Plant cells are also now available as hosts, and control sequences
compatible with plant cells such as the nopaline synthase promoter and
polyadenylation signal sequences (Depicker, A., et al., J. Mol. Appl. Gen.
(1982) 1:561) are available.
Recently, in addition, expression systems employing insect cells utilizing
the control systems provided by baculovirus vectors have been described
(Miller, D. W., et al., in Genetic Engineering (1986) Setlow, J. K. et
al., eds., Plenum Publishing, Vol. 8, pp. 277-297). These systems are also
successful in producing Taq polymerase.
Transformations
Depending on the host cell used, transformation is done using standard
techniques appropriate to such cells. The calcium treatment employing
calcium chloride, as described by Cohen, S. N., Proc. Natl. Acad. Sci.
(USA) (1972) 69:2110 is used for procaryotes or other cells that contain
substantial cell wall barriers. Infection with Agrobacterium tumefaciens
(Shaw, C. H., et al., Gene (1983) 23:315) is used for certain plant cells.
For mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology (1978) 52:546 is
preferred. Transformations into yeast are carried out according to the
method of Van Solingen, P., et al., J. Bact. (1977) 130:946 and Hsiao, C.
L., et al., Proc. Natl. Acad. Sci. (USA) (1979) 76:3829.
Construction of a .lambda.gt11 Expression Library
The strategy for isolating DNA encoding desired proteins such as the Taq
polymerase encoding DNA, using the bacteriophage vector lambda gt11, is as
follows. A library can be constructed of EcoRI-flanked AluI fragments,
generated by complete digestion of Thermus aquaticus DNA, inserted at the
EcoRI site in the lambda gt11 phage (Young and Davis, Proc. Natl. Acad.
Sci USA (1983) 80:1194-1198). Because the unique EcoRI site in this
bacteriophage is located in the carboxyl-terminus of the
.beta.-galactosidase gene, inserted DNA (in the appropriate frame and
orientation) is expressed as protein fused with .beta.-galactosidase under
the control of the lactose operon prompter/operator.
Genomic expression libraries are then screened using the antibody plaque
hybridization procedure. A modification of this procedure, referred to as
"epitope selection," uses antiserum against the fusion protein sequence
encoded by the phage, to confirm the identification of hybridized plaques.
Thus, this library of recombinant phages could be screened with antibodies
that recognize the 86,000-90,000 dalton Taq polymerase in order to
identify phage that carry DNA segments encoding the antigenic determinants
of this protein.
Approximately 2.times.10.sup.5 recombinant phage are screened using total
rabbit Taq polymerase antiserum. In this primary. screen, positive signals
are detected and one or more of these plaques are purified from candidate
plaques which failed to react with preimmune serum and reacted with immune
serum and analyzed in some detail. To examine the fusion proteins produced
by the recombinant phage, lysogens of the phage in the host Y1089 are
produced. Upon induction of the lysogens and gel electrophoresis of the
resulting proteins, each lysogen may be observed to produce a new protein,
not found in the other lysogens, or duplicate sequences may result. Phage
containing positive signals are picked., in this case, one positive plaque
was picked for further identification and replated at lower densities to
purify recombinants and the purified clones were analyzed by size class
via digestion with EcoRI restriction enzyme. Probes can then be made of
the isolated DNA insert sequences and labeled appropriately and these
probes can be used in conventional colony or plaque hybridization assays
described in Maniatis et al., Molecular Cloning: A Laboratory Manual
(1982), the disclosure of which is incorporated herein by reference.
The labeled probe was used to probe a second genomic library constructed in
a Charon 35 bacteriophage (Wilhelmine, A. M. et al., Gene (1983)
26:171-179). This library was made from Sau3A partial digestions of
genomic Thermus aquaticus DNA and size fractionated fragments (15-20 kb)
were cloned into the BamHI site of the Charon 35 phage. The probe was used
to isolate phage containing DNA encoding the Taq polymerase. One of the
resulting phage, designated CH35:Taq#4-2, was found to contain the entire
gene sequence. Partial sequences encoding portions of the gene were also
isolated.
Vector Construction
Construction of suitable vectors containing the desired coding and control
sequences employs standard ligation and restriction techniques that are
well understood in the art. Isolated plasmids, DNA sequences, or
synthesized oligonucleotides are cleaved, tailored, and religated in the
form desired.
Site-specific DNA cleavage is performed by treating with the suitable
restriction enzyme (or enzymes) under conditions that are generally
understood in the art, and the particulars of which are specified by the
manufacturer of these commercially available restriction enzymes. See,
e.g., New England Biolabs, Product Catalog. In general, about 1 .mu.g of
plasmid or DNA sequence is cleaved by one unit of enzyme in about 20 .mu.l
of buffer solution; in the examples herein, typically an excess of
restriction enzyme is used to ensure complete digestion of the DNA
substrate. Incubation times of about one hour to two hours at about
37.degree. C. are workable, although variations can be tolerated. After
each incubation, protein is removed by extraction with phenol/chloroform,
and may be followed by o ether extraction, and the nucleic acid recovered
from aqueous fractions by precipitation with ethanol. If desired, size
separation of the cleaved fragments may be performed by polyacrylamide gel
or agarose gel electrophoresis using standard techniques. A general
description of size separations is found in Methods in Enzymology (1980)
65:499-560.
Restriction-cleaved fragments may be blunt-ended by treating with the large
fragment of E. coli DNA polymerase I (Klenow) in the presence of the four
deoxynucleotide triphosphates (dNTPs) using incubation times of about 15
to 25 minutes at 20 to 25.degree. C. in 50 mM Tris pH 7.6, 50 mM NaCl, 10
mM MgCl.sub.2, 10 mM DTT and 50-100 .mu.M dNTPs. The Klenow fragment fills
in at 5' sticky ends, but chews back protruding 3' single strands, even
though the four dNTPs are present. If desired, selective repair can be
performed by supplying only one of the, or selected, dNTPs within the
limitations dictated by the nature of the sticky ends. After treatment
with Klenow, the mixture is extracted with phenol/chloroform and ethanol
precipitated. Treatment under appropriate conditions with S1 nuclease
results in hydrolysis of any single-stranded portion.
Synthetic oligonucleotides may be prepared using the triester method of
Matteucci, et al., (J. Am. Chem. Soc. (1981) 103:3185-3191) or using
automated synthesis methods. Kinasing of single strands prior to annealing
or for labeling is achieved using an excess, e.g., approximately 10 units
of polynucleotide kinase to 1 nM substrate in the presence of 50 mM Tris,
pH 7.6, 10 mM MgCl.sub.2, 5 mM dithiothreitol, 1-2 mM ATP. If kinasing is
for labeling of probe, the ATP will contain high specific activity
.gamma.-.sup.32 P.
Ligations are performed in 15-30 .mu.l volumes under the following standard
conditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl.sub.2, 10 mM
DTT, 33 .mu.g/ml BSA, 10 mM-50 mM NaCl, and either 40 .mu.M ATP, 0.01-0.02
(Weiss) units T4 DNA ligase at 0.degree. C. (for "sticky end" ligation) or
1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt
end" ligation). Intermolecular "sticky end" ligations are usually
performed at 33-100 .mu.g/ml total DNA concentrations (5-100 nM total end
concentration). Intermolecular blunt end ligations (usually employing a
10-30 fold molar excess of linkers) are performed at 1 .mu.M total ends
concentration.
In vector construction employing "vector fragments", the vector fragment is
commonly treated with bacterial alkaline phosphatase (BAP) in order to
remove the 5' phosphate and prevent religation of the vector. BAP
digestions are conducted at pH 8 in approximately 150 mM Tris, in the
presence of Na.sup.+ and Mg.sup.+ 2 using about 1 unit of BAP per mg of
vector at 60.degree. C. for about one hour. In order to recover the
nucleic acid fragments, the preparation is extracted with
phenol/chloroform and ethanol precipitated. Alternatively, religation can
be prevented in vectors that have been double digested by additional
restriction enzyme digestion of the unwanted fragments.
Modification of DNA Sequences
For portions of vectors derived from cDNA or genomic DNA that require
sequence modifications, site-specific primer-directed mutagenesis is used.
This technique is now standard in the art, and is conducted using a primer
synthetic oligonucleotide complementary to a single-stranded phage DNA to
be mutagenized except for limited mismatching, representing the desired
mutation. Briefly, the synthetic oligonucleotide is used as a primer to
direct synthesis of a strand complementary to the phage, and the resulting
double-stranded DNA is transformed into a phage-supporting host bacterium.
Cultures of the transformed bacteria are plated in top agar, permitting
plaque formation from single cells that harbor the phage.
Theoretically, 50% of the new plaques will contain the phage having, as a
single strand, the mutated form; 50% will have the original sequence. The
plaques are transferred to nitrocellulose filters and the "lifts"
hybridized with kinased synthetic primer at a temperature that permits
hybridization of an exact match, but at which the mismatches with the
original strand are | | |
