U.S. patent application number 10/060432 was filed with the patent office on 2003-02-27 for transaminases and aminotranferases.
This patent application is currently assigned to Diversa Corporation. Invention is credited to Swanson, Ronald V., Warren, Patrick V..
Application Number | 20030040092 10/060432 |
Document ID | / |
Family ID | 24398535 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030040092 |
Kind Code |
A1 |
Warren, Patrick V. ; et
al. |
February 27, 2003 |
Transaminases and aminotranferases
Abstract
Thermostable transaminase and aminotransferase enzymes derived
from various ammonifex, aquifex and pyrobaculum organisms are
disclosed. The enzymes are produced from native or recombinant host
cells and can be utilized in the pharmaceutical, agricultural and
other industries.
Inventors: |
Warren, Patrick V.; (San
Diego, CA) ; Swanson, Ronald V.; (Del Mar,
CA) |
Correspondence
Address: |
FISH & RICHARDSON, PC
4350 LA JOLLA VILLAGE DRIVE
SUITE 500
SAN DIEGO
CA
92122
US
|
Assignee: |
Diversa Corporation
|
Family ID: |
24398535 |
Appl. No.: |
10/060432 |
Filed: |
January 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10060432 |
Jan 29, 2002 |
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09481733 |
Jan 11, 2000 |
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09481733 |
Jan 11, 2000 |
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09069226 |
Apr 27, 1998 |
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6013509 |
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09069226 |
Apr 27, 1998 |
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08599171 |
Feb 9, 1996 |
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5814473 |
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Current U.S.
Class: |
435/193 ;
435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/1096
20130101 |
Class at
Publication: |
435/193 ;
435/69.1; 435/320.1; 435/325; 536/23.2 |
International
Class: |
C12N 009/10; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
We claim:
1. An isolated polynucleotide having at least 70% identity to a
polynucleotide encoding an enzyme having aminotransferase and/or
transaminase activity, wherein said polynucleotide is amplified
using an oligonucleotide primer pair selected from:
3 5' CCGAGAATTCATTAAAGAGGAGAAATTAACTATGATTGAAGACCCTATGGAC (SEQ. ID
NO:1) and 3' CGAAGATCTTTAGCACTTCTCTCAGGTTC; (SEQ. ID NO:2) 5'
CCGAGAATTCATTAAAGAGGAGAAATTAACTATGGACAGGCTTGAAAAAGTA (SEQ ID NO:3)
and 3' CGGAAGATCTTCAGCTAAGCTTCTCTAAGAA; (SEQ ID NO:4) 5'
CCGACAATTGATTAAAGAGGAGAAATTAACTATGTGGGAATTAGA- CCCTAAA (SEQ ID
NO:5) and 3' CGGAGGATCCCTACACCTCTTTTTCAAGCT; (SEQ ID NO:6) 5'
CCGACAATTGATTAAAGAGGAGAAATTAACTATGACATAC- TTAATGAACAAT (SEQ ID
NO:7) and 3' CGGAAGATCTTTATGAGAAGTCCCTT- TCAAG; (SEQ ID NO:8) 5'
CCGAGAATTCATTAAAGAGGAGAAATTAACTATG- CGGAAACTGGCCGAGCGG (SEQ ID
NO:9) and 3' CGGAGGATCCTTAAAGTGCCGCTTCGATCAA; (SEQ ID NO:10) 5'
CCGACAATTGATTAAAGAGGAGAAATTAACTATGTGCGGGATAGTCGGATAC (SEQ ID NO:11)
and 3' CGGAAGATCTTTATTCCACCGTGACCGTTTT; (SEQ ID NO:12) 5'
CCGACAATTGATTAAAGAGGAGAAATTAACTATGATACCCCAGAGGATTAAG (SEQ ID NO:13)
and 3' CGGAAGATCTTTAAAGAGAGCTTGAAAGGGA; (SEQ ID NO:14) 5'
CCGAGAATTCATTAAAGAGGAGAAATTAACIATGAAGCCGTACGCTA- AATAT (SEQ ID
NO:15) and 3' CGGAAGATCTCTAATACACAGGAGTGATCCA; (SEQ ID NO:16) 5'
CCGAGAATTCATTAGAGGAGAAATTAACTATGGCAGTCA- AAGTGCGGCCT (SEQ ID NO:33)
and 3' -CGGAGGATCCTTATCCAAAGCTTCC- AGGAAG; (SEQ ID NO:34) or 5'
CCGAGAATTCATTAAAGAGGAGAAATTAACTATGAGAAAAGGACTTGCAAGT (SEQ ID NO:37)
and 3' CGGAGGATCCTTAGATCTCTTCAAGGGCTTT. (SEQ ID NO:38)
Description
[0001] This application is a Continuation of U.S. patent
application Ser. No. 08/599,171 filed on Feb. 9, 1996.
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, the use of such
polynucleotides and polypeptides, as well as the production and
isolation of such polynucleotides and polypeptides. More
particularly, the polynucleotides and polypeptides of the present
invention have been putatively identified as transaminases and/or
aminotransferases. Aminotransferases are enzymes that catalyze the
transfer of amino groups from .alpha.-amino to .alpha.-keto acids.
They are also called transaminases.
[0003] The .alpha.-amino groups of the 20 L-amino acids commonly
found in proteins are removed during the oxidative degradation of
the amino acids. The removal of the .alpha.-amino groups, the first
step in the catabolism of most of the L-amino acids, is promoted by
aminotransferases (or transaminases). In these transamination
reactions, the .alpha.-amino group is transferred to the
.alpha.-carbon atom of .alpha.-ketoglutarate, leaving behind the
corresponding .alpha.-keto acid analog of the amino acid. There is
no net deamination (i.e., loss of amino groups) in such reactions
because the .alpha.-ketoglutarate becomes aminated as the
.alpha.-amino acid is deaminated. The effect of transamination
reactions is to collect the amino groups from many different amino
acids in the form of only one, namely, L-glutamate. The glutamate
channels amino groups either into biosynthetic pathways or into a
final sequence of reactions by which nitrogenous waste products are
formed and then excreted.
[0004] Cells contain several different aminotransferases, many
specific for .alpha.-ketoglutarate as the amino group acceptor. The
aminotransferases differ in their specificity for the other
substrate, the L-amino acid that donates the amino group, and are
named for the amino group donor. The reactions catalyzed by the
aminotransferases are freely reversible, having an equilibrium
constant of about 1.0 (.DELTA.G.sup.0'.apprxeq.0 kJ/mol).
[0005] Aminotransferases are classic examples of enzymes catalyzing
bimolecular ping-pong reactions. In such reactions the first
substrate must leave the active site before the second substrate
can bind. Thus the incoming amino acid binds to the active site,
donates its amino group to pyridoxal phosphate, and departs in the
form of an .alpha.-keto acid. Then the incoming .alpha.-keto acid
is bound, accepts the amino group from pyridoxamine phosphate, and
departs in the form of an amino acid.
[0006] The measurement of alanine aminotransferase and aspartate
aminotransferase levels in blood serum is an important diagnostic
procedure in medicine, used as an indicator of heart damage and to
monitor recovery from the damage.
[0007] The polynucleotides and polypeptides of the present
invention have been identified as transaminases and/or
aminotransferases as a result of their enzymatic activity.
[0008] In accordance with one aspect of the present invention,
there are provided novel enzymes, as well as active fragments,
analogs and derivatives thereof.
[0009] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules encoding the
enzymes of the present invention including mRNAs, cDNAs, genomic
DNAs as well as active analogs and fragments of such enzymes.
[0010] In accordance with another aspect of the present invention
there are provided isolated nucleic acid molecules encoding mature
polypeptides expressed by the DNA contained in ATCC Deposit No.
______.
[0011] In accordance with yet a further aspect of the present
invention, there is provided a process for producing such
polypeptides by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a
nucleic acid sequence of the present invention, under conditions
promoting expression of said enzymes and subsequent recovery of
said enzymes.
[0012] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such enzymes,
or polynucleotides encoding such enzymes for transferring an amino
group from an .alpha.-amino acid to an .alpha.-keto acid. Most
transaminases use L-amino acids as substrates, but as described
below, it is also possible to convert the transaminases of the
invention to use D-amino acids as substrates, thereby increasing
their array of uses to include, for example, manufacture of
synthetic pyrethroids and as components of .beta.-lactam
antibiotics. The transaminases of the invention are stable at high
temperatures and in organic solvents and, thus, are superior for
use with L- and/or D-amino acids for production of optically pure
chiral compounds used in pharmaceutical, agricultural and other
chemical industries.
[0013] In accordance with yet a further aspect of the present
invention, there are also provided nucleic acid probes comprising
nucleic acid molecules of sufficient length to hybridize to a
nucleic acid sequence of the present invention.
[0014] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such enzymes,
or polynucleotides encoding such enzymes, for in vitro purposes
related to scientific research, for example, to generate probes for
identifying similar sequences which might encode similar enzymes
from other organisms by using certain regions, i.e., conserved
sequence regions, of the nucleotide sequence.
[0015] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
[0016] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0017] FIG. 1 is an illustration of the full-length DNA (SEQ ID
NO:17) and corresponding deduced amino acid sequence (SEQ ID NO:25)
of Aquifex aspartate transaminase A of the present invention.
Sequencing was performed using a 378 automated DNA sequencer
(Applied Biosystems, Inc.) for all sequences of the present
invention.
[0018] FIG. 2 is an illustration of the full-length DNA (SEQ ID
NO:18) and corresponding deduced amino acid sequence (SEQ ID NO:26)
of Aquifex aspartate aminotransferase B.
[0019] FIG. 3 is an illustration of the full-length DNA (SEQ ID
NO:19) and corresponding deduced amino acid sequence (SEQ ID NO:27)
of Aquifex adenosyl-8-amino-7-oxononanoate aminotransferase.
[0020] FIG. 4 is an illustration of the full-length DNA (SEQ ID
NO:20) and corresponding deduced amino acid sequence (SEQ ID NO:28)
of Aquifex acetylornithine aminotransferase.
[0021] FIG. 5 is an illustration of the full-length DNA (SEQ ID
NO:21) and corresponding deduced amino acid sequence (SEQ ID NO:29)
of Ammonifex degensii aspartate aminotransferase.
[0022] FIG. 6 is an illustration of the full-length DNA (SEQ ID
NO:22) and corresponding deduced amino acid sequence (SEQ ID NO:30)
of Aquifex glucosamine:fructose-6-phosphate aminotransferase.
[0023] FIG. 7 is an illustration of the full-length DNA (SEQ ID
NO:23) and corresponding deduced amino acid sequence (SEQ ID NO:31)
of Aquifex histidinol-phosphate aminotransferase.
[0024] FIG. 8 is an illustration of the full-length DNA (SEQ ID
NO:24) and corresponding deduced amino acid sequence (SEQ ID NO:32)
of Pyrobacullum aerophilum branched chain aminotransferase.
[0025] FIG. 9 is a diagramatic illustration of the assay used to
assess aminotransferase activity of the proteins using glutamate
dehydrogenase.
[0026] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0027] A coding sequence is "operably linked to" another coding
sequence when RNA polymerase will transcribe the two coding
sequences into a single mRNA, which is then translated into a
single polypeptide having amino acids derived from both coding
sequences. The coding sequences need not be contiguous to one
another so long as the expressed sequences ultimately process to
produce the desired protein.
[0028] "Recombinant" enzymes refer to enzymes produced by
recombinant DNA techniques; i.e., produced from cells transformed
by an exogenous DNA construct encoding the desired enzyme.
"Synthetic" enzymes are those prepared by chemical synthesis.
[0029] A DNA "coding sequence of" or a "nucleotide sequence
encoding" a particular enzyme, is a DNA sequence which is
transcribed and translated into an enzyme when placed under the
control of appropriate regulatory sequences.
[0030] In accordance with an aspect of the present invention, there
are provided isolated nucleic acids (polynucleotides) which encode
for the mature enzymes having the deduced amino acid sequences of
FIGS. 1-8 (SEQ ID NOS:17-32).
[0031] In accordance with another aspect of the present invention,
there are provided isolated polynucleotides encoding the enzymes of
the present invention. The deposited material is a mixture of
genomic clones comprising DNA encoding an enzyme of the present
invention. Each genomic clone comprising the respective DNA has
been inserted into a pQE vector (Quiagen, Inc., Chatsworth,
Calif.). The deposit has been deposited with: the American Type
Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852,
USA, on Dec. 13, 1995 and assigned ATCC Deposit No. ______.
[0032] The deposit(s) have been made under the terms of the
Budapest Treaty on the International Recognition of the deposit of
micro-organisms for purposes of patent procedure. The strains will
be irrevocably and without restriction or condition released to the
public upon the issuance of a patent. These deposits are provided
merely as convenience to those of skill in the art and are not an
admission that a deposit would be required under 35 U.S.C.
.sctn.112. The sequences of the polynucleotides contained in the
deposited materials, as well as the amino acid sequences of the
polypeptides encoded thereby, are controlling in the event of any
conflict with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and no such
license is hereby granted.
[0033] The polynucleotides of this invention were originally
recovered from genomic DNA libraries derived from the following
organisms:
[0034] Aquifex VF5 is a Eubacteria which was isolated in Vulcano,
Italy. It is a gram-negative, rod-shaped, strictly
chemolithoautotrophic, marine organism which grows optimally at
85-90.degree. C. (T.sub.max=95.degree. C.) at pH 6.8 in a high salt
culture medium with Q as a substrate, and H.sub.2/CO.sub.2+0.5%
O.sub.2 in gas phase.
[0035] Ammonifex degensii KC4 is a new Eubacaterial organism
isolated in Java, Indonesia. This Gram negative chemolithoautotroph
has three respiration systems. The bacterium can utilize nitrate,
sulfate, and sulfur. The organism grows optimally at 70.degree. C.,
and pH 7.0, in a low salt culture medium with 0.2% nitrate as a
substrate and H.sub.2/CO.sub.2 in gas phase.
[0036] Pyrobaculum aerophilium IM2 is a thermophilic sulfur archaea
(Crenarchaeota) isolated in Ischia Maronti, Italy. It is a
rod-shaped organism that grows optimally at 100.degree. C. at pH
7.0 in a low salt culture medium with nitrate, yeast extract,
peptone, and O.sub.2 as substrates and N.sub.2/CO.sub.2, O.sub.2 in
gas phase.
[0037] Accordingly, the polynucleotides and enzymes encoded thereby
are identified by the organism from which they were isolated, and
are sometimes hereinafter referred to as "VF5/ATA" (FIG. 1 and SEQ
ID NOS:17 and 25), "VF5/AAB" (FIG. 2 and SEQ ID NOS:18 and 26),
"VF5/A87A" (FIG. 3 and SEQ ID NOS:19 and 27), "VF5/AOA" (FIG. 4 and
SEQ ID NOS:20 and 28), "KC4/AA" (FIG. 5 and SEQ ID NOS:21 and 29),
"VF5/GF6PA" (FIG. 6 and SEQ ID NOS:22 and 30), "VF5/HPA" (FIG. 7
and SEQ ID NOS:23 and 31) and "IM2/BCA" (FIG. 8 and SEQ ID NOS:24
and 32).
[0038] The polynucleotides and polypeptides of the present
invention show identity at the nucleotide and protein level to
known genes and proteins encoded thereby as shown in Table 1.
1TABLE 1 Protein Protein DNA Gene w/closet Similarity Identity
Identity Enzyme Homology (Organism) (%) (%) (%) VF5/ATA Bacillus
subtilis 57.5 38.3 50.1 VF5/AAB Sulfolobus solfataricus 62.5 33.0
50.1 VF5/A87A Bacillus sphaericus BioA 67.4 42.9 51 VF5/AOA
Bacillus subtilis argD 70.6 48.7 52.0 KC4/AA Bacillus YM-2 aspC
72.6 52.7 52.0 VF5/GF6PA Rhizobium 66.3 47.7 51.0 Leguminosarum
NodM VF5/HPA Bacillus subtilis 55.7 32.6 45.3 HisH/E. coli HisC
(same gene) IM2/BCA E. coli iluE 63.7 43.6 49.7
[0039] All the clones identified in Table 1 encode polypeptides
which have transaminase or aminotransferase activity.
[0040] One means for isolating the nucleic acid molecules encoding
the enzymes of the present invention is to probe a gene library
with a natural or artificially designed probe using art recognized
procedures (see, for example: Current Protocols in Molecular
Biology, Ausubel F. M. et al. (EDS.) Green Publishing Company
Assoc. and John Wiley Interscience, New York, 1989, 1992). It is
appreciated by one skilled in the art that the polynucleotides of
SEQ ID NOS:17-24, or fragments thereof (comprising at least 12
contiguous nucleotides), are particularly useful probes. Other
particularly useful probes for this purpose are hybridizable
fragments of the sequences of SEQ ID NOS:1-9 (i.e., comprising at
least 12 contiguous nucleotides).
[0041] With respect to nucleic acid sequences which hybridize to
specific nucleic acid sequences disclosed herein, hybridization may
be carried out under conditions of reduced stringency, medium
stringency or even stringent conditions. As an example of
oligonucleotide hybridization, a polymer membrane containing
immobilized denatured nucleic acids is first prehybridized for 30
minutes at 45.degree. C. in a solution consisting of 0.9 M NaCl, 50
mM NaH.sub.2PO.sub.4, pH 7.0, 5.0 mM Na.sub.2 EDTA, 0.5% SDS,
10.times. Denhardt's, and 0.5 mg/mL polyriboadenylic acid.
Approximately 2.times.10.sup.7 cpm (specific activity
4-9.times.10.sup.8 cpm/ug) of .sup.32P end-labeled oligonucleotide
probe are then added to the solution. After 12-16 hours of
incubation, the membrane is washed for 30 minutes at room
temperature in 1.times. SET (150 mM NaCl, 20 mM Tris hydrochloride,
pH 7.8, 1 mM Na.sub.2EDTA) containing 0.5% SDS, followed by a 30
minute wash in fresh 1.times. SET at Tm-10.degree. C. (Tm is minus
10.degree. C.) for the oligo-nucleotide probe. The membrane is then
exposed to auto-radiographic film for detection of hybridization
signals.
[0042] Stringent conditions means hybridization will occur only if
there is at least 90% identity, preferably at least 95% identity
and most preferably at least 97% identity between the sequences.
See J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d
Ed., Cold Spring Harbor Laboratory (1989) which is hereby
incorporated by reference in its entirety.
[0043] As used herein, a first DNA (RNA) sequence is at least 70%
and preferably at least 80% identical to another DNA (RNA) sequence
if there is at least 70% and preferably at least a 80% or 90%
identity, respectively, between the bases of the first sequence and
the bases of the another sequence, when properly aligned with each
other, for example when aligned by BLASTN.
[0044] The present invention relates to polynucleotides which
differ from the reference polynucleotide such that the changes are
silent changes, for example the change does not or the changes do
not alter the amino acid sequence encoded by the polynucleotide.
The present invention also relates to nucleotide changes which
result in amino acid substitutions, additions, deletions, fusions
and truncations in the polypeptide encoded by the reference
polynucleotide. In a preferred aspect of the invention these
polypeptides retain the same biological action as the polypeptide
encoded by the reference polynucleotide.
[0045] The polynucleotides of this invention were recovered from
genomic gene libraries from the organisms listed in Table 1. Gene
libraries were generated in the Lambda ZAP II cloning vector
(Stratagene Cloning Systems). Mass excisions were performed on
these libraries to generate libraries in the pBluescript phagemid.
Libraries were generated and excisions were performed according to
the protocols/methods hereinafter described.
[0046] The polynucleotides of the present invention may be in the
form of RNA or DNA which DNA includes cDNA, genomic DNA, and
synthetic DNA. The DNA may be double-stranded or single-stranded,
and if single stranded may be the coding strand or non-coding
(anti-sense) strand. The coding sequences which encodes the mature
enzymes may be identical to the coding sequences shown in FIGS. 1-8
(SEQ ID NOS:17-24) or may be a different coding sequence which
coding sequence, as a result of the redundancy or degeneracy of the
genetic code, encodes the same mature enzymes as the DNA of FIGS.
1-8 (SEQ ID NOS:17-24).
[0047] The polynucleotide which encodes for the mature enzyme of
FIGS. 1-8 (SEQ ID NOS:25-32) may include, but is not limited to:
only the coding sequence for the mature enzyme; the coding sequence
for the mature enzyme and additional coding sequence such as a
leader sequence or a proprotein sequence; the coding sequence for
the mature enzyme (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequence 5'
and/or 3' of the coding sequence for the mature enzyme.
[0048] Thus, the term "polynucleotide encoding an enzyme (protein)"
encompasses a polynucleotide which includes only coding sequence
for the enzyme as well as a polynucleotide which includes
additional coding and/or non-coding sequence.
[0049] The present invention further relates to variants of the
hereinabove described polynucleotides which encode for fragments,
analogs and derivatives of the enzymes having the deduced amino
acid sequences of FIGS. 1-8 (SEQ ID NOS;25-32). The variant of the
polynucleotide may be a naturally occurring allelic variant of the
polynucleotide or a non-naturally occurring variant of the
polynucleotide.
[0050] Thus, the present invention includes polynucleotides
encoding the same mature enzymes as shown in FIGS. 1-8 (SEQ ID
NOS:17-24) as well as variants of such polyynucleotides which
variants encode for a fragment, derivative or analog of the enzymes
of FIGS. 1-8 (SEQ ID NOS:17-24). Such nucleotide variants include
deletion variants, substitution variants and addition or insertion
variants.
[0051] As hereinabove indicated, the polynucleotides may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequences shown in FIGS. 1-8 (SEQ ID NOS:17-24). As
known in the art, an allelic variant is an alternate form of a
polynucleotide sequence which may have a substitution, deletion or
addition of one or more nucleotides, which does not substantially
alter the function of the encoded enzyme. Also, using directed and
other evolution strategies, one may make very minor changes in DNA
sequence which can result in major changes in function.
[0052] Fragments of the full length gene of the present invention
may be used as hybridization probes for a cDNA or a genomic library
to isolate the full length DNA and to isolate other DNAs which have
a high sequence similarity to the gene or similar biological
activity. Probes of this type preferably have at least 10,
preferably at least 15, and even more preferably at least 30 bases
and may contain, for example, at least 50 or more bases. The probe
may also be used to identify a DNA clone corresponding to a full
length transcript and a genomic clone or clones that contain the
complete gene including regulatory and promotor regions, exons and
introns. An example of a screen comprises isolating the coding
region of the gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary or identical to that of the gene or portion of the
gene sequences of the present invention are used to screen a
library of genomic DNA to determine which members of the library
the probe hybridizes to.
[0053] It is also appreciated that such probes can be and are
preferably labeled with an analytically detectable reagent to
facilitate identification of the probe. Useful reagents include but
are not limited to radioactivity, fluorescent dyes or enzymes
capable of catalyzing the formation of a detectable product. The
probes are thus useful to isolate complementary copies of DNA from
other sources or to screen such sources for related sequences.
[0054] The present invention further relates to polynucleotides
which hybridize to the hereinabove-described sequences if there is
at least 70%, preferably at least 90%, and more preferably at least
95% identity between the sequences. The present invention
particularly relates to polynucleotides which hybridize under
stringent conditions to the hereinabove-described polynucleotides.
As herein used, the term "stringent conditions" means hybridization
will occur only if there is at least 95% and preferably at least
97% identity between the sequences. The polynucleotides which
hybridize to the hereinabove described polynucleotides in a
preferred embodiment encode enzymes which either retain
substantially the same biological function or activity as the
mature enzyme encoded by the DNA of FIGS. 1-8 (SEQ ID
NOS:17-24).
[0055] Alternatively, the polynucleotide may have at least 15
bases, preferably at least 30 bases, and more preferably at least
50 bases which hybridize to any part of a polynucleotide of the
present invention and which has an identity thereto, as hereinabove
described, and which may or may not retain activity. For example,
such polynucleotides may be employed as probes for the
polynucleotides of SEQ ID NOS:17-24, for example, for recovery of
the polynucleotide or as a diagnostic probe or as a PCR primer.
[0056] Thus, the present invention is directed to polynucleotides
having at least a 70% identity, preferably at least 90% identity
and more preferably at least a 95% identity to a polynucleotide
which encodes the enzymes of SEQ ID NOS:25-32 as well as fragments
thereof, which fragments have at least 15 bases, preferably at
least 30 bases and most preferably at least 50 bases, which
fragments are at least 90% identical, preferably at least 95%
identical and most preferably at least 97% identical under
stringent conditions to any portion of a polynucleotide of the
present invention.
[0057] The present invention further relates to enzymes which have
the deduced amino acid sequences of FIGS. 1-8 (SEQ ID NOS:17-24) as
well as fragments, analogs and derivatives of such enzyme.
[0058] The terms "fragment," "derivative" and "analog" when
referring to the enzymes of FIGS. 1-8 (SEQ ID NOS:25-32) means
enzymes which retain essentially the same biological function or
activity as such enzymes. Thus, an analog includes a proprotein
which can be activated by cleavage of the proprotein portion to
produce an active mature enzyme.
[0059] The enzymes of the present invention may be a recombinant
enzyme, a natural enzyme or a synthetic enzyme, preferably a
recombinant enzyme.
[0060] The fragment, derivative or analog of the enzymes of FIGS.
1-8 (SEQ ID NOS:25-32) may be (i) one in which one or more of the
amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code, or (ii) one in which one or more
of the amino acid residues includes a substituent group, or (iii)
one in which the mature enzyme is fused with another compound, such
as a compound to increase the half-life of the enzyme (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature enzyme, such as a leader or secretory
sequence or a sequence which is employed for purification of the
mature enzyme or a proprotein sequence. Such fragments, derivatives
and analogs are deemed to be within the scope of those skilled in
the art from the teachings herein.
[0061] The enzymes and polynucleotides of the present invention are
preferably provided in an isolated form, and preferably are
purified to homogeneity.
[0062] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or enzyme present in a living animal is not
isolated, but the same polynucleotide or enzyme, separated from
some or all of the coexisting materials in the natural system, is
isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or enzymes could be part of a composition, and
still be isolated in that such vector or composition is not part of
its natural environment.
[0063] The enzymes of the present invention include the enzymes of
SEQ ID NOS:25-32 (in particular the mature enzyme) as well as
enzymes which have at least 70% similarity (preferably at least 70%
identity) to the enzymes of SEQ ID NOS:25-32 and more preferably at
least 90 % similarity (more preferably at least 90% identity) to
the enzymes of SEQ ID NOS:25-32 and still more preferably at least
95% similarity (still more preferably at least 95% identity) to the
enzymes of SEQ ID NOS:25-32 and also include portions of such
enzymes with such portion of the enzyme generally containing at
least 30 amino acids and more preferably at least 50 amino
acids.
[0064] As known in the art "similarity" between two enzymes is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one enzyme to the sequence of a second
enzyme.
[0065] A variant, i.e. a "fragment", "analog" or "derivative"
polypeptide, and reference polypeptide may differ in amino acid
sequence by one or more substitutions, additions, deletions,
fusions and truncations, which may be present in any
combination.
[0066] Among preferred variants are those that vary from a
reference by conservative amino acid substitutions. Such
substitutions are those that substitute a given amino acid in a
polypeptide by another amino acid of like characteristics.
Typically seen as conservative substitutions are the replacements,
one for another, among the aliphatic amino acids Ala, Val, Leu and
Ile; interchange of the hydroxyl residues Ser and Thr, exchange of
the acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0067] Most highly preferred are variants which retain the same
biological function and activity as the reference polypeptide from
which it varies.
[0068] Fragments or portions of the enzymes of the present
invention may be employed for producing the corresponding
full-length enzyme by peptide synthesis; therefore, the fragments
may be employed as intermediates for producing the full-length
enzymes. Fragments or portions of the polynucleotides of the
present invention may be used to synthesize full-length
polynucleotides of the present invention.
[0069] The present invention also relates to vectors which include
polynucleotides of the present invention, host cells which are
genetically engineered with vectors of the invention and the
production of enzymes of the invention by recombinant
techniques.
[0070] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector such as an expression
vector. The vector may be, for example, in the form of a plasmid, a
phage, etc. The engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating
promoters, selecting transformants or amplifying the genes of the
present invention. The culture conditions, such as temperature, pH
and the like, are those previously used with the host cell selected
for expression, and will be apparent to the ordinarily skilled
artisan.
[0071] The polynucleotides of the present invention may be employed
for producing enzymes by recombinant techniques. Thus, for example,
the polynucleotide may be included in any one of a variety of
expression vectors for expressing an enzyme. Such vectors include
chromosomal, nonchromosomal and synthetic DNA sequences, e.g.,
derivatives of SV40; bacterial plasmids; phage DNA; baculovirus;
yeast plasmids; vectors derived from combinations of plasmids and
phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus,
and pseudorabies. However, any other vector may be used as long as
it is replicable and viable in the host.
[0072] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0073] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. Zac or tip, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0074] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0075] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the protein.
[0076] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Bacillus subtilis; fungal cells, such as yeast; insect cells such
as Drosophila S2 and Spodoptera SJ9; animal cells such as CHO, COS
or Bowes melanoma; adenoviruses; plant cells, etc. The selection of
an appropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
[0077] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBluescript II
KS, ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic:
pXT1, pSG5 (Stratagene) pSVK3, pBPV, pMSG, pSVL SV40 (Pharmacia).
However, any other plasmid or vector may be used as long as they
are replicable and viable in the host.
[0078] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lac, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L and trp. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0079] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0080] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the enzymes of the invention can be
synthetically produced by conventional peptide synthesizers.
[0081] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0082] Transcription of the DNA encoding the enzymes of the present
invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0083] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated enzyme. Optionally, the
heterologous sequence can encode a fusion enzyme including an
N-terminal identification peptide imparting desired
characteristics, e.g., stabilization or simplified purification of
expressed recombinant product.
[0084] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0085] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0086] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0087] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0088] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well known to those skilled in the art.
[0089] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0090] The enzyme can be recovered and purified from recombinant
cell cultures by methods including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0091] The enzymes of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the enzymes of the present
invention may be glycosylated or may be non-glycosylated. Enzymes
of the invention may or may not also include an initial methionine
amino acid residue.
[0092] Transaminases are a group of key enzymes in the metabolism
of amino acids and amino sugars and are found in all organisms from
microbes to mammals. In the transamination reaction, an amino group
is transferred from an amino acid to an .alpha.-keto acid.
Pyridoxal phosphate is required as a co-factor to mediate the
transfer of the amino group without liberation of ammonia.
[0093] Amino acids currently have applications as additives to
aminal feed, human nutritional supplements, components in infusion
solutions, and synthetic intermediates for manufacture of
pharmaceuticals and agricultural products. For example, L-glutamic
acid is best known as a flavor enhancer for human food. L-lysine
and L-methionine are large volume additives to animal feed and
human supplements. L-tryptophan and L-threonine have similar
potential applications. L-phenylalanine and L-aspartic acid have
very important market potential as key components in the
manufacture of the low-calorie sweetener aspartame, and other
promising low-calorie sweeteners have compositions containing
certain amino acids as well. Infusion solutions require a large
range of amino acids including those essential ones in human
diets.
[0094] Transaminases are highly stereoselective, and most use
L-amino acids as substrates. Using the approach disclosed in a
commonly assigned, copending provisional application Serial No.
60/008,316, filed on Dec. 7, 1995 and entitled "Combinatorial
Enzyme Development," the disclosure of which is incorporated herein
by reference in its entirety, one can convert the transaminases of
the invention to use D-amino acids as substrates. Such conversion
makes possible a broader array of transaminase applications. For
instance, D-valine can be used in the manufacture of synthetic
pyrethroids. D-phenylglycine and its derivatives can be useful as
components of .beta.-lactam antibiotics. Further, the thermostable
transaminases have superior stability at higher temperatures and in
organic solvents. Thus, they are better suited to utilize either L-
and/or D-amino acids for production of optically pure chiral
compounds used in pharmaceutical, agricultural, and other chemical
manufactures.
[0095] There are a number of reasons to employ transaminases in
industrial-scale production of amino acids and their
derivatives.
[0096] 1) Transaminases can catalyze stereoselective synthesis of
D- or L-amino acids from their corresponding .alpha.-keto acids.
Therefore no L- or D-isomers are produced, and no resolution is
required.
[0097] 2) Tramminases have uniformly high catalytic rates, capable
of converting up to 400 .mu.moles of substrates per minute per mg
enzyme.
[0098] 3) Many required .alpha.-keto acids can be conveniently
prepared by chemical synthesis at low cost.
[0099] 4) The capital investment for an immobilized enzyme process
using transaminases is much lower than for a large scale
fermentation process, and productivity of the bioreactor is often
an order of magnitude higher.
[0100] 5) The technology is generally applicable to a broad range
of D- or L-amino acids because transaminases exist with varying
specificities. Such broad scope allows a number of different L- or
D-amino acids to be produced with the same equipment and often the
same biocatalyst.
[0101] Antibodies generated against the enzymes corresponding to a
sequence of the present invention can be obtained by direct
injection of the enzymes into an animal or by administering the
enzymes to an animal, preferably a nonhuman. The antibody so
obtained will then bind the enzymes itself. In this manner, even a
sequence encoding only a fragment of the enzymes can be used to
generate antibodies binding the whole native enzymes. Such
antibodies can then be used to isolate the enzyme from cells
expressing that enzyme.
[0102] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, Nature, 256:495-497, 1975), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., Immunology Today
4:72, 1983), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole et al., in Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985).
[0103] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic enzyme products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic enzyme products of this invention.
[0104] Antibodies generated against an enzyme of the present
invention may be used in screening for similar enzymes from other
organisms and samples. Such screening techniques are known in the
art, for example, one such screening assay is described in Sambrook
and Maniatis, Molecular Cloning: A Laboratory Manual (2d Ed.), vol.
2:Section 8.49, Cold Spring Harbor Laboratory, 1989, which is
hereby incorporated by reference in its entirety.
[0105] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
[0106] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0107] "Plasmids" are designated by a lower case "p" preceded
and/or followed by capital letters and/or numbers. The starting
plasmids herein are either commercially available, publicly
available on an unrestricted basis, or can be constructed from
available plasmids in accord with published procedures. In
addition, equivalent plasmids to those described are known in the
art and will be apparent to the ordinarily skilled artisan.
[0108] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a
polyacrylamide gel to isolate the desired fragment.
[0109] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel et al., Nucleic
Acids Res., 8:4057 (1980).
[0110] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0111] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T., et al., Id., p. 146). Unless otherwise provided, ligation may
be accomplished using known buffers and conditions with 10 units of
T4 DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
[0112] Unless otherwise stated, transformation was performed as
described in Sambrook and Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1989.
EXAMPLE 1
Bacterial Expression and Purification of Transaminases and
Aminotransferases
[0113] DNA encoding the enzymes of the present invention, SEQ ID
NOS:25 through 32, were initially amplified from a pBluescript
vector containing the DNA by the PCR technique using the primers
noted herein. The amplified sequences were then inserted into the
respective PQE vector listed beneath the primer sequences, and the
enzyme was expressed according to the protocols set forth herein.
The genomic DNA has also been used as a template for the PCR
amplification, i.e., once a positive clone has been identified and
primer sequences determined using the cDNA, it was then possible to
return to the genomic DNA and directly amplify the desired
sequence(s) there. The 5' and 3' primer sequences and the vector
for the respective genes are as follows:
2 Aquifex Aspartate Transaminase A aspa501 5'
CCGAGAATTCATTAAAGAGGAGAAATTAACTATGATGAAGACCCTATGGAC (SEQ. ID NO:1)
aspa301 3' CGAAGATCTTTAGCACTTCTCTCAGGTTC (SEQ. ID NO:2) vector:
pQET1 Aquifex Aspartate Aminotransferase B aspb501 5'
CCGAGAATTCATTAAAGAGGAGAAATTAACTATGGACAGGCTTGAAAAAGTA (SEQ ID NO:3)
aspb301 3' CGGAAGATCTTCAGCTAAGCTTCTCTAAGAA (SEQ ID NO:4) vector:
pQET1 Aquifex Adenosyl-8-amino-7-oxononanoat- e Aminotransferase
ameth501 5' CCGACAATTGATAAGAGGAGAAATTAACTATGTGGG- AATTAGACCCTAAA
(SEQ ID NO:5) ameth301 3' CGGAGGATCCCTACACCTCTTTTTC- AAGCT (SEQ ID
NO:6) vector: pQET12 Aquifex Acetylornithine Aminotransferase aorn
501 5' CCGACAATTGATTAAAGAGGAGAAATTAACTATGACATACTTAATGAACAAT (SEQ ID
NO:7) aorn 301 3' CGGAAGATCTTTATGAGAAGTCCCTTTCAAG (SEQ ID NO:8)
vector: pQET12 Ammonifex desgensii Aspartate Aminotransferase adasp
501 5' CCGAGAATTCATTAAAGAGGAGAAATTAACTATGCGGAAACTGGCCGAGCGG (SEQ ID
NO:9) adasp 301 3' CGGAGGATCCTTAAAGTGCCGCTTCGATCAA (SEQ ID NO:10)
vector: pQET12 Aquifex Glucosamine:Fructose-6-phosphate
Aminotransferase glut 501 5'
CCGACAATTGATTAAAGAGGAGAAATTAACTATGTGCGGGATAGTCGGATAC (SEQ ID NO:11)
glut 301 3' CGGAAGATCTTTATTCCACCGTGACCGTTTT (SEQ ID NO:12) vector:
pQET1 Aquifex Histadine-phosphate Aminotransferase his 501 5'
CCGACAATTGATTAAAGAGGAGAAATTAACTATGATAC- CCCAGAGGATTAAG (SEQ ID
NO:13) his 301 3' CGGAAGATCTTTAAAGAGAGCTTGA- AAGGGA (SEQ ID NO:14)
vector: pQET1 Pyrobacullum aerophilum Branched Chain
Aminotransferase bcat 501 5'
CCGAGAATTCATTAAAGAGGAGAAATTAACTATGAAGCCGTACGCTAAATAT (SEQ ID NO:15)
bcat 301 3' CGGAAGATCTCTAATACACAGGAGTGATCCA (SEQ ID NO:16) vector:
pQET1
[0114] The restriction enzyme sites indicated correspond to the
restriction enzyme sites on the bacterial expression vector
indicated for the respective gene (Qiagen, Inc. Chatsworth,
Calif.). The pQE vector encodes antibiotic resistance (Amp.sup.r),
a bacterial origin of replication (ori), an IPTG-regulatable
promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag
and restriction enzyme sites.
[0115] The pQE vector was digested with the restriction enzymes
indicated. The amplified sequences were ligated into the respective
pQE vector and inserted in frame with the sequence encoding for the
RBS. The ligation mixture was then used to transform the E. coli
strain M15/pREP4 (Qiagen, Inc.) by electroporation. M15/pREP4
contains multiple copies of the plasmid pREP4, which expresses the
lacI repressor and also confers kanamycin resistance (Kan.sup.r).
Transformants were identified by their ability to grow on LB plates
and ampicillin/kanamycin resistant colonies were selected. Plasmid
DNA was isolated and confirmed by restriction analysis. Clones
containing the desired constructs were grown overnight (O/N) in
liquid culture in LB media supplemented with both Amp (100 ug/ml)
and Kan (25 ug/ml). The O/N culture was used to inoculate a large
culture at a ratio of 1:100 to 1:250. The cells were grown to an
optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-thiogalacto pyranoside") was then added to a fmal
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene expression.
Cells were grown an extra 3 to 4 hours. Cells were then harvested
by centrifugation.
[0116] The primer sequences set out above may also be employed to
isolate the target gene from the deposited material by
hybridization techniques described above.
EXAMPLE 2
Isolation of a Selected Clone from the Deposited Genomic Clones
[0117] The two oligonucleotide primers corresponding to the gene of
interest are used to amplify the gene from the deposited material.
A polymerase chain reaction is carried out in 25 .mu.l of reaction
mixture with 0.1 .mu.g of the DNA of the gene of interest. The
reaction mixture is 1.5-5 mM MgCl.sub.2, 0.01 % (w/v) gelatin, 20
.mu.M each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and
1.25 Unit of Taq polymerase. Thirty cycles of PCR (denaturation at
94.degree. C. for 1 min; annealing at 55.degree. C. for 1 min;
elongation at 72.degree. C. for 1 min) are performed with the
Perkin-Elmer Cetus 9600 thermal cycler. The amplified product is
analyzed by agarose gel electrophoresis and the DNA band with
expected molecular weight is excised and purified. The PCR product
is verified to be the gene of interest by subcloning and sequencing
the DNA product.
[0118] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the invention
may be practiced otherwise than as particularly described.
Sequence CWU 1
1
* * * * *