U.S. patent application number 10/027805 was filed with the patent office on 2002-11-07 for esterases.
This patent application is currently assigned to Diversa Corporation. Invention is credited to Kosmatka, Anna, Link, Steven, Maffia, Anthony, Murphy, Dennis, Reid, John, Robertson, Dan E., Swanson, Ronald V., Warren, Patrick V..
Application Number | 20020164725 10/027805 |
Document ID | / |
Family ID | 32108491 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164725 |
Kind Code |
A1 |
Robertson, Dan E. ; et
al. |
November 7, 2002 |
Esterases
Abstract
Esterase enzymes derived from various Staphylothermus,
Pyrodictium, Archaeoglobus, Aquifex, M11TL, Thermococcus,
Teredinibacter and Sulfolobus organisms are disclosed. The enzymes
are produced from native or recombinant host cells and can be
utilized in pharmaceutical, agricultural and other industries.
Inventors: |
Robertson, Dan E.; (Solana
Beach, NJ) ; Murphy, Dennis; (Malvern, PA) ;
Reid, John; (Armore, PA) ; Maffia, Anthony;
(Wilmington, DE) ; Link, Steven; (Wilmington,
DE) ; Swanson, Ronald V.; (Del Mar, CA) ;
Warren, Patrick V.; (Coatesville, PA) ; Kosmatka,
Anna; (Doylestown, PA) |
Correspondence
Address: |
GARY CARY WARE & FRIENDENRICH LLP
4365 EXECUTIVE DRIVE
SUITE 1600
SAN DIEGO
CA
92121-2189
US
|
Assignee: |
Diversa Corporation
San Diego
CA
|
Family ID: |
32108491 |
Appl. No.: |
10/027805 |
Filed: |
December 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10027805 |
Dec 21, 2001 |
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09903410 |
Jul 10, 2001 |
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09903410 |
Jul 10, 2001 |
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09382242 |
Aug 24, 1999 |
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09382242 |
Aug 24, 1999 |
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08602359 |
Feb 16, 1996 |
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5942430 |
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Current U.S.
Class: |
435/106 |
Current CPC
Class: |
C12P 13/04 20130101;
C12N 9/18 20130101; A61K 38/00 20130101; A01K 2217/05 20130101;
C12N 9/16 20130101 |
Class at
Publication: |
435/106 |
International
Class: |
C12P 013/04 |
Claims
What is claimed is:
1. A method for transferring an amino group from an amino acid to
an .alpha.-keto acid comprising: Contacting an amino acid in the
presence of an .alpha.-keto acid with an enzyme selected from the
group consisting of an enzyme having the amino acid sequence set
forth in SEQ ID NOS:33-42.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of co-pending U.S. patent
application Ser. No. 08/602,359, filed Feb. 17, 1996.
SPECIFICATION
[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 esterases. Esterases
are enzymes that catalyze the hydrolysis of ester groups to organic
acids and alcohols.
[0003] Many esterases are known and have been discovered in a broad
variety of organisms, including bacteria, yeast and higher animals
and plants. A principal example of esterases are the lipases, which
are used in the hydrolysis of lipids, acidolysis(replacement of an
esterified fatty acid with a free fatty acid) reactions,
transesterification(exchang- e of fatty acids between
triglycerides)reactions, and in ester synthesis. The major
industrial applications for lipases include: the detergent
industry, where they are employed to decompose fatty materials in
laundry stains into easily removable hydrophilic substances; the
food and beverage industry where they are used in the manufacture
of cheese, the ripening and flavoring of cheese, as antistaling
agents for bakery products, and in the production of margarine and
other spreads with natural butter flavors; in waste systems; and in
the pharmaceutical industry where they are used as digestive
aids.
[0004] The polynucleotides and polypeptides of the present
invention have been identified as esterases as a result of their
enzymatic activity.
[0005] In accordance with one aspect of the present invention,
there are provided novel enzymes, as well as active fragments,
analogs and derivatives thereof.
[0006] 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.
[0007] 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.______.
[0008] 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.
[0009] 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 hydrolyzing ester
groups to yield an organic acid and an alcohol. The esterases of
the invention are stable at high temperatures and in organic
solvents and, thus, are superior for use in production of optically
pure chiral compounds used in pharmaceutical, agricultural and
other chemical industries.
[0010] 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.
[0011] 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.
[0012] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
[0013] 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.
[0014] FIG. 1 is an illustration of the full-length DNA (SEQ ID
NO:23) and corresponding deduced amino acid sequence (SEQ ID NO:33)
of Staphylothebnus marinus F1-12LC of the present invention.
Sequencing was performed using a 378 automated DNA sequencer
(Applied Biosystems, Inc.) for all sequences of the present
invention.
[0015] FIG. 2 is an illustration of the full-length DNA (SEQ ID
NO:24) and corresponding deduced amino acid sequence (SEQ ID NO:34)
of Pyrodictium TAG11-17LC.
[0016] FIG. 3 is an illustration of the full-length DNA (SEQ ID
NO:25) and corresponding deduced amino acid sequence (SEQ ID NO:35)
of Archaeoglobus venificus SNP6-24LC.
[0017] FIG. 4 is an illustration of the full-length DNA (SEQ ID
NO:26) and corresponding deduced amino acid sequence (SEQ ID NO:36)
of Aquifex pyrophilus-28LC.
[0018] FIG. 5 is an illustration of the full-length DNA (SEQ ID
NO:27) and corresponding deduced amino acid sequence (SEQ ID NO:37)
of M11TL-29L.
[0019] FIG. 6 is an illustration of the full-length DNA (SEQ ID
NO:28) and corresponding deduced amino acid sequence (SEQ ID NO:38)
of Thermococcus CL-2-30LC.
[0020] FIG. 7 is an illustration of the full-length DNA (SEQ ID
NO:29) and corresponding deduced amino acid sequence (SEQ ID NO:39)
of Aquifex VF5-34LC.
[0021] FIG. 8 is an illustration of the full-length DNA (SEQ ID
NO:30) and corresponding deduced amino acid sequence (SEQ ID NO:40)
of Teredinibacter-42L.
[0022] FIG. 9 is an illustration of the full-length DNA (SEQ ID
NO:31) and corresponding deduced amino acid sequence (SEQ ID NO:41)
of Archaeoglobus fulgidus VC16-16MC.
[0023] FIG. 10 is an illustration of the full-length DNA (SEQ ID
NO:32) and corresponding deduced amino acid sequence (SEQ ID NO:42)
of Sulfolobus solfataricus P1-8LC.
[0024] 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).
[0025] 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.
[0026] "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.
[0027] 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.
[0028] 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. 1-10 (SEQ ID NOS:23-32).
[0029] 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 pBluescript vector (Stratagene, La Jolla,
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. ______.
[0030] 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.
[0031] The polynucleotides of this invention were originally
recovered from genomic gene libraries derived from the following
organisms:
[0032] Staphylothermus marinus F1 is a thermophilic sulfur archaea
which was isolated in Vulcano, Italy. It grows optimally at
85.degree. C. (T.sub.max 98.degree. C.) at pH 6.5.
[0033] Pyrodictium TAG11 is a thermophilic sulfur archaea which was
isolated in the Middle Atlantic Ridge. It grows optimally at
103.degree. C. (T.sub.max=110.degree. C.) at pH 6.5.
[0034] Archaeoglobus venificus SNP6 was isolated in the Middle
Atlantic Ridge and grows optimally at 75.degree. C.
(T.sub.max=92.degree. C.) at pH 6.9.
[0035] Aquifex pyrophilus K01 5a was isolated at Kolbeinsey Ridge,
North of Iceland. This marine organism is a gram-negative,
rod-shaped, strictly chemolithoautrophic, knall gas bacterium. It
grows optimally at 85.degree. C. (T.sub.max=95.degree. C.) at pH
6.8.
[0036] M11TL is a new species of Desulfurococcus which was isolated
from Diamond Pool (formerly Jim's Black Pool) in Yellowstone. The
organism grows heterotrophically by fermentation of different
organic materials (sulfur is not necessary) in grape-like
aggregates optimally at 85-88.degree. C. in a low salt medium at pH
7.0.
[0037] Thermococcus CL-2 was isolated in the North Cleft Segment of
the Juan de Fuca Ridge from a severed alvinellid worm residing on a
"black smoker" sulfide structure. This marine archaea forms
pleomorphic cocci, and grows optimally at 88.degree. C.
[0038] Aquifex VF5 was isolated at a beach in Vulcano, Italy. This
marine organism is a gram-negative, rod-shaped, strictly
chemolithoautotrophic, knall gas bacterium. It grows optimally at
85.degree. C. (T.sub.max=95.degree. C.) at pH 6.8.
[0039] Teredinibacter (pure) is an endosymbiont of the shipworm
Bankia gouldi. The organism has straight to slightly bent 5-10
.mu.m rods, and forms spiral cells as stationary phase is met. The
organism was described in Science (1983) 22:1401-1403. It grows
optimally at 30.degree. C. at pH 8.0.
[0040] Archaeoglobus fulgidus VC16 was isolated in Vulcano, Italy.
The organism grows optimally at 85.degree. C. (T.sub.max=92.degree.
C.) at pH 7.0.
[0041] Sulfolobus solfataricus P1 grows optimally at 85.degree. C.
(T.sub.max=87.degree. C.) at pH 2.0.
[0042] Accordingly, the polynucleotides and enzymes encoded thereby
are identified by the organism from which they were isolated, and
are sometimes hereinafter referred to as F1/12LC (FIG. 1 and SEQ ID
NOS:23 and 33), TAG11/17LC (FIG. 2 and SEQ ID NOS:24 and 34),
SNP6/24LC (FIG. 3 and SEQ ID NOS:25 and 35), AqP/28LC (FIG. 4 and
SEQ ID NOS:26 and 36), M11TL/29L (FIG. 5 and SEQ ID NOS:27 and 37),
CL-2/30LC (FIG. 6 and SEQ ID NOS:28 and 38), VF5/34LC (FIG. 7 and
SEQ ID NOS:29 and 39), Trb/42L (FIG. 8 and SEQ ID NOS:30 and 40),
VC16/16MC (FIG. 9 and SEQ ID NOS:31 and 41) and P1/8LC (FIG. 10 and
SEQ ID NOS: 32 and 42).
[0043] 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/closest Similarity Identity
Identity Enzyme Homology (Organism) (%) (%) (%) F1/12LC No
significant homology -- -- -- TAG11/17LC No significant homology --
-- -- SNP6/24LC PIR S34609 - 46 27 42 carboxylesterase Pseudomones
sp. (strain KWI-56) open reading frame of unknown function in
E.coli. AqP/29LC 53 31 38 M11TL/29LC No significant homology -- --
-- CL02/30LC No significant homology -- -- -- VF5/34LC Identified
by homology 84 71 71 to 28LC; also homologous to ORF of unknown
function 5' of tgs in E. coli Trb/42L No significant homology -- --
-- P1-8LC VC16-16MC
[0044] All the clones identified in Table 1 encode polypeptides
which have esterase activity.
[0045] This invention, in addition to the isolated nucleic acid
molecules encoding the enzymes of the present invention, also
provides substantially similar sequences. Isolated nucleic acid
sequences are substantially similar if: (i) they are capable of
hybridizing under conditions hereinafter described, to the
polynucleotides of SEQ ID NOS:23-32; (ii) or they encode DNA
sequences which are degenerate to the polynucleotides of SEQ ID
NOS:23-32. Degenerate DNA sequences encode the amino acid sequences
of SEQ ID NOS:33-42, but have variations in the nucleotide coding
sequences. As used herein, substantially similar refers to the
sequences having similar identity to the sequences of the instant
invention. The nucleotide sequences that are substantially the same
can be identified by hybridization or by sequence comparison.
Enzyme sequences that are substantially the same can be identified
by one or more of the following: proteolytic digestion, gel
electrophoresis and/or microsequencing.
[0046] 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, N.Y., 1989, 1992). It is
appreciated by one skilled in the art that the polynucleotides of
SEQ ID NOS:23-32, 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: 1-22 (i.e., comprising at
least 12 contiguous nucleotides).
[0047] 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.2EDTA, 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. for the
oligo-nucleotide probe. The membrane is then exposed to
auto-radiographic film for detection of hybridization signals.
[0048] 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.
[0049] 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 lest 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.
[0050] The present invention relates to polynucleotides which
differ from the reference polynucleotide such that the changes are
silent changes, for example the change 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.
[0051] 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.
[0052] 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.
1-10 (SEQ ID NOS:23-32) 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.
1-10 (SEQ ID NOS:23-32).
[0053] The polynucleotide which encodes for the mature enzyme of
FIGS. 1-10 (SEQ ID NOS:33-42) 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.
[0054] 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.
[0055] 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. 1-10 (SEQ ID NOS:33-42). The variant of the
polynucleotide may be a naturally occurring allelic variant of the
polynucleotide or a non-naturally occurring variant of the
polynucleotide.
[0056] Thus, the present invention includes polynucleotides
encoding the same mature enzymes as shown in FIGS. 1-10 (SEQ ID
NOS:23-32) as well as variants of such polynucleotides which
variants encode for a fragment, derivative or analog of the enzymes
of FIGS. 1-10 (SEQ ID NOS:23-32). Such nucleotide variants include
deletion variants, substitution variants and addition or insertion
variants.
[0057] As hereinabove indicated, the polynucleotides may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequences shown in FIGS. 1-10 (SEQ ID NOS:23-32). 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.
[0058] 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 to that of the gene of the present invention are used
to screen a library of genomic DNA to determine which members of
the library the probe hybridizes to.
[0059] 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.
[0060] 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. 1-10 (SEQ ID
NOS:23-32).
[0061] 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:23-32, for example, for recovery of
the polynucleotide or as a diagnostic probe or as a PCR primer.
[0062] 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:33-42 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.
[0063] The present invention further relates to enzymes which have
the deduced amino acid sequences of FIGS. 1-10 (SEQ ID NOS:23-32)
as well as fragments, analogs and derivatives of such enzyme.
[0064] The terms "fragment," "derivative" and "analog" when
referring to the enzymes of FIGS. 1-10 (SEQ ID NOS:33-42) mean
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.
[0065] The enzymes of the present invention may be a recombinant
enzyme, a natural enzyme or a synthetic enzyme, preferably a
recombinant enzyme.
[0066] The fragment, derivative or analog of the enzymes of FIGS.
1-10 (SEQ ID NOS:33-42) 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.
[0067] The enzymes and polynucleotides of the present invention are
preferably provided in an isolated form, and preferably are
purified to homogeneity.
[0068] 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.
[0069] The enzymes of the present invention include the enzymes of
SEQ ID NOS:33-42 (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:33-42 and more preferably at
least 90% similarity (more preferably at least 90% identity) to the
enzymes of SEQ ID NOS:33-42 and still more preferably at least 95%
similarity (still more preferably at least 95% identity) to the
enzymes of SEQ ID NOS:33-42 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.
[0070] 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.
[0071] 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.
[0072] 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
lle; 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.
[0073] Most highly preferred are variants which retain the same
biological function and activity as the reference polypeptide from
which it varies.
[0074] 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.
[0075] 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.
[0076] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, 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.
[0077] 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.
[0078] 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.
[0079] 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. lac or trp, 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.
[0080] 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.
[0081] 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.
[0082] 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 Sf9; 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.
[0083] 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.
[0084] 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 lacl, 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.
[0085] 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)).
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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 GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0092] 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.
[0093] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0094] 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.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] Esterases are a group of key enzymes in the metabolism of
fats and are found in all organisms from microbes to mammals. In
the hydrolysis reaction, an ester group is hydrolysed to an organic
acid and an alcohol.
[0099] Esterases enantiomerically differentiate dicarboxylic
diesters and diacetates of diols. Using the approach disclosed in a
commonly assigned, copending provisional application Ser. 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 could convert the
enantiospecificity of the esterase. Further, the thermostable
esterases are believed to have superior stability at higher
temperatures and in organic solvents. Thus, they are better suited
for use in rigorous production proceeds which require robust
catalysts.
[0100] There are a number of industrial and scientific applications
for esterases, such as those of the present invention,
including:
[0101] 1) Esterases are useful in the dairy industry as ripening
starters for cheeses, such as the Swiss-type cheeses;
[0102] 2) Esterases are useful in the pulp and paper industry for
lignin removal from cellulose pulps, for lignin solubilization by
cleaving the ester linkages between aromatic acids and lignin and
between lignin and hemicelluloses, and for disruption of cell wall
structure when used in combination with xylanase and other
xylan-degrading enzymes in biopulping and biobleaching of
pulps;
[0103] 3) Esterases are useful in the synthesis of carbohydrate
derivatives, such as sugar derivatives;
[0104] 4) Esterases are useful, when combined with xylanases and
cellulases, in the conversion of lignocellulosic wastes to
fermentable sugars for producing a variety of chemicals and
fuels;
[0105] 5) Esterases are useful as research reagents in studies on
plant cell wall structure, particularly the nature of covalent
bonds between lignin and carbohydrate polymers in the cell wall
matrix;
[0106] 6) Esterases are also useful as research reagents in studies
on mechanisms related to disease resistance in plants and the
process of organic matter decomposition; and
[0107] 7) Esterases are useful in selection of plants bred for
production of highly digestible animal feeds, particularly for
ruminant animals.
[0108] 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.
[0109] 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).
[0110] 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.
[0111] 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
et al., Molecular Cloning: A Laboratory Manual (2d Ed.), Cold
Spring Harbor Laboratory, Section 12.21-12.28 (1989) which is
hereby incorporated by reference in its entirety.
[0112] 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.
[0113] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0114] "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.
[0115] "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 82 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.
[0116] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel et al., Nucleic
Acids Res., 8:4057 (1980).
[0117] "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.
[0118] "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.
[0119] Unless otherwise stated, transformation was performed as
described in Sambrook et al., Molecular Cloning: A Laboratory
Manual (2d Ed.), Cold Spring Harbor Press (1989).
EXAMPLE 1
Bacterial Expression and Purification of Esterases
[0120] DNA encoding the enzymes of the present invention, SEQ ID
NOS:33 through 42, 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 5' and 3' primer sequences for the respective genes are as
follows:
2 Staphylothermus marinus F1-12LC 5' CCGAGAATTC ATTAAAGAGG
AGAAATTAAC TATGTCTTTA AACAAGCACT CT 3' CGGAAGATCT CTATCGTTTA
GTGTATGATT T vector: pQET Pyrodictium TAG11-17LC 5' CCGAGAATTC
ATTAAAGAGG AGAAATTAAC TATGAAACTC CTTGAGCCCA CA EcoRI 3' CGGAAGATCT
CGCCGGTACA CCATCAGCCA C BglII vector: pQET Archaeoglobus venifficus
SNP6-24LC 5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCCATAT GTTAGGAATG
GT 3' CGGAGGTACC TTAGAACTGT GCTGAAGAAA TAAATTCGTC CATTGCTCT 3'
CGGAGGTACC TTAGAACTGT GCTGAAGAAA TAAATTCGTC CATTGCTCTA TTA vector:
pQET Aquifex pyrophilus - 28LC 5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC
TATGAGATTG AGGAAATTTG AAG 3' CGGAGGTACC CTATTCAGAA AGTACCTCTA A
vector: pQET M11TL - 29LC 5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC
TATGTTTAAT ATCAATGTCT TT 3' CGGAAGATCT TTAAGGATTT TCCCTGGGTA G
vector: pQET Thermococcus CL-2 - 30LC 5' CCGAGAATTC ATTAAAGAGG
AGAAATTAAC TATGGAGGTT TACAAGGCCA AA 3' CGGAGGTACC TTATTGAGCC
GAAGAGTACG A vector: pQET Aquifex VF5 - 34LC 5' CCGAGAATTC
ATTAAAGAGG AGAAATTAAC TATGATTGGC AATTTGAAAT TGA EcoRI 3' CGGAGGTACC
TTAAAGTGCT CTCATATCCC C KpnI vector: pQET Teredinibacter 42L 5'
CCGAGAATTC ATTAAAGACG AGAAATTAAC TATGCCAGCT AATGACTCAC CC 3'
CGGAAGATCT TCAACAGGCT CCAAATAATT TC (without His-tag) 3' CGGAAGATCT
ACAGGCTCCA AATAATTTC (with His-tag) vector: pQE12 Archaeoglobus
fulgidus VC16-16MC 5' CCGAGAATTC ATTAAAGAGG AGAAATTAAC TATGCTTGAT
ATGCCAATCG AC EcoRl 3' CGGAGGTACC CTAGTCGAAG ACAAGAAGAG C Kpn1
vector: pQET Sulfolabus solfataricus P1-8LC 5' CCGAGAATTC
ATTAAAGAGG AGAAATTAAC TATGCCCCAG GATCCTAGAA TT EcoRl 3' CGGAGGTACC
TTAAATTTTA TCATAAAATA C Kpn1 vector: pQET
[0121] 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.
[0122] 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
lacl 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 final
concentration of 1 mM. IPTG induces by inactivating the lacl
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.
[0123] 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
[0124] 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.
EXAMPLE 3
Production of the Expression Gene Bank
[0125] Colonies containing pBluescript plasmids with random inserts
from the organisms M11TL, Thermococcus GU5L5, and Teredinibacter
were obtained according to the method of Hay and Short, Strategies,
5:16, 1992.
EXAMPLE 4
Screening for Lipase/Esterase Activity
[0126] The resulting colonies were picked with sterile toothpicks
and used to singly inoculate each of the wells of 96-well
microtiter plates. The wells contained 250 .mu.L of LB media with
100 .mu.g/mL ampicillin, 80 .mu.g/mL methicillin, and 10% v/v
glycerol (LB Amp/Meth, glycerol). The cells were grown overnight at
37.degree. C. without shaking. This constituted generation of the
"Source GeneBank." Each well of the Source GeneBank thus contained
a stock culture of E. coli cells, each of which contained a
pBluescript with a unique DNA insert.
[0127] The plates of the Source GeneBank were used to multiply
inoculate a single plate (the "Condensed Plate") containing in each
well 200 .mu.L of LB Amp/Meth, glycerol. This step was performed
using the High Density Replicating Tool (HDRT) of the Beckman
Biomek with a 1% bleach, water, isopropanol, air-dry sterilization
cycle in between each inoculation. Each well of the Condensed Plate
thus contained 10 to 12 different pBluescript clones from each of
the source library plates. The Condensed Plate was grown for 16
hours at 37.degree. C. and then used to inoculate two white 96-well
Polyfiltronics microtiter daughter plates containing in each well
250 1.mu.L of LB Amp/Meth (no glycerol). The original condensed
plate was put in storage -80.degree. C. The two condensed daughter
plates were incubated at 37.degree. C. for 18 hours.
[0128] The short chain esterase `600 .mu.M substrate stock
solution` was prepared as follows: 25 mg of each of the following
compounds was dissolved in the appropriate volume of DMSO to yield
a 25.2 mM solution. The compounds used were 4-methylumbelliferyl
proprionoate, 4-methylumbelliferyl butyrate, and
4-methylumbelliferyl heptanoate. Two hundred fifty microliters of
each DMSO solution was added to ca 9 mL of 50 mM, pH 7.5 Hepes
buffer which contained 0.6% of Triton X-100 and 0.6 mg per mL of
dodecyl maltoside (Anatrace). The volume was taken to 10.5 mL with
the above Hepes buffer to yield a slightly cloudy suspension.
[0129] The long chain `600 .mu.M substrate stock solution` was
prepared as follows: 25 mg of each of the following compounds was
dissolved in DMSO to 25.2 mM as above. The compounds used were
4-methylumbelliferyl elaidate, 4-methylumbelliferyl palmitate,
4-methylumbelliferyl oleate, and 4-methylumbelliferyl stearate. All
required brief warming in a 70.degree. C. bath to achieve
dissolution. Two hundred fifty microliters of each DMSO solution
was added to the Hepes buffer and diluted to 10.5 mL as above. All
seven umbelliferones were obtained from Sigma Chemical Co.
[0130] Fifty .mu.L of the long chain esterase or short chain
esterase `600 .mu.M substrate stock solution` was added to each of
the wells of a white condensed plate using the Biomek to yield a
final concentration of substrate of about 100 .mu.M. The
fluorescence values were recorded (excitation=326 nm, emission=450
nm) on a plate-reading fluorometer immediately after addition of
the substrate. The plate was incubated at 70.degree. C. for 60
minutes in the case of the long chain substrates, and 30 minutes at
RT in the case of the short chain substrates. The fluorescence
values were recorded again. The initial and final fluorescence
values were compared to determine if an active clone was
present.
EXAMPLE 5
Isolation and Purification of the Active Clone
[0131] To isolate the individual clone which carried the activity,
the Source GeneBank plates were thawed and the individual wells
used to singly inoculate a new plate containing LB Amp/Meth. As
above, the plate was incubated at 37.degree. C. to grow the cells,
50 .mu.L of 600 .mu.M substrate stock solution was added using the
Biomek and the fluorescence was determined. Once the active well
from the source plate was identified, cells from this active well
were streaked on agar with LB/Amp/Meth and grown overnight at
37.degree. C. to obtain single colonies. Eight single colonies were
picked with a sterile toothpick and used to singly inoculate the
wells of a 96-well microtiter plate. The wells contained 250 .mu.L
of LB Amp/Meth. The cells were grown overnight at 37.degree. C.
without shaking. A 200 .mu.L aliquot was removed from each well and
assayed with the appropriate long or short chain substrates as
above. The most active clone was identified and the remaining 50
.mu.L of culture was used to streak an agar plate with LB/Amp/Meth.
Eight single colonies were picked, grown and assayed as above. The
most active clone was used to inoculate 3 mL cultures of
LB/Amp/Meth, which were grown overnight. The plasmid DNA was
isolated from the cultures and utilized for sequencing.
[0132] 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.
* * * * *