U.S. patent application number 13/107465 was filed with the patent office on 2011-12-15 for substantially pure reverse transcriptases and methods of production thereof.
This patent application is currently assigned to LIFE TECHNOLOGIES, INC.. Invention is credited to A. John Hughes, JR..
Application Number | 20110306112 13/107465 |
Document ID | / |
Family ID | 28677874 |
Filed Date | 2011-12-15 |
United States Patent
Application |
20110306112 |
Kind Code |
A1 |
Hughes, JR.; A. John |
December 15, 2011 |
SUBSTANTIALLY PURE REVERSE TRANSCRIPTASES AND METHODS OF PRODUCTION
THEREOF
Abstract
The present invention provides substantially pure reverse
transcriptases, which are preferably substantially free from
contamination with nucleic acids. The invention also provides
methods for the production of these enzymes, and kits comprising
these enzymes which may be used in synthesizing, amplifying or
sequencing nucleic acid molecules, including through the use of the
polymerase chain reaction, particularly RT-PCR.
Inventors: |
Hughes, JR.; A. John;
(Temecula, CA) |
Assignee: |
LIFE TECHNOLOGIES, INC.
Carlsbad
CA
|
Family ID: |
28677874 |
Appl. No.: |
13/107465 |
Filed: |
May 13, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12243887 |
Oct 1, 2008 |
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13107465 |
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11212807 |
Aug 29, 2005 |
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12243887 |
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10640662 |
Aug 14, 2003 |
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11212807 |
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09533548 |
Mar 23, 2000 |
6630333 |
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10640662 |
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60126050 |
Mar 23, 1999 |
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Current U.S.
Class: |
435/194 |
Current CPC
Class: |
C12N 9/1241 20130101;
C12N 9/1276 20130101 |
Class at
Publication: |
435/194 |
International
Class: |
C12N 9/12 20060101
C12N009/12 |
Claims
1. A reverse transcriptase purified by a method comprising
permeabilizing a cellular source of reverse transcriptase,
subjecting said permeabilized cellular source of reverse
transcriptase to filtration, and isolating said reverse
transcriptase, wherein said reverse transcriptase is substantially
free of nucleic acids.
2. The reverse transcriptase of claim 1, wherein said cellular
source is a bacterial cell or a recombinant bacterial cell.
3. The reverse transcriptase of claim 2, wherein said
permeabilization forms spheroplasts and/or protoplasts.
4. The reverse transcriptase of claim 1, wherein said filtration
comprises microfiltration or ultrafiltration.
5. The reverse transcriptase of claim 1, wherein said
permeabilization comprises contacting said cellular source with an
aqueous solution comprising a chaotropic agent or a nonionic
detergent.
6. The reverse transcriptase of claim 5, wherein said nonionic
detergent is Triton X-100 or sodium deoxycholate.
7. The reverse transcriptase of claim 1, wherein said isolating
comprises column chromatography.
8. The reverse transcriptase of claim 1, wherein said method is
conducted under conditions favoring the partitioning of nucleic
acids from said reverse transcriptase.
9. The reverse transcriptase of claim 8, wherein said conditions
comprise microfiltration of spheroplasts or protoplasts in the
presence of ammonium sulfate.
10. The reverse transcriptase of claim 1, wherein said reverse
transcriptase is MMLV RT or MMLV RT substantially reduced in RNase
H activity.
11. The reverse transcriptase of claim 5, wherein said chaotropic
agent is guanidine, urea or guanidine hydrochloride.
12. A reverse transcriptase substantially free of nucleic
acids.
13. The reverse transcriptase of claim 12, wherein said reverse
transcriptase is MMLV RT or MMLV H-RT.
14. The reverse transcriptase of claim 1, wherein said reverse
transcriptase is RSV H-RT, AMV H-RT, RAV H-RT, MAV RT or HIV
H-RT.
15. The reverse transcriptase of claim 12, wherein said reverse
transcriptase is RSV H-RT, AMV H-RT, RAV H-RT, MAV RT or HIV H-RT.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 10/640,662, filed Aug. 14, 2003, which is a
divisional of U.S. application Ser. No. 09/533,548, filed Mar. 23,
2000, now U.S. Pat. No. 6,630,333, which claims the benefit of U.S.
Provisional Patent Application No. 60/126,050, filed Mar. 23, 1999,
the disclosures of each of which are incorporated herein by
reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention is in the fields of molecular biology,
protein chemistry and protein purification. Specifically, the
invention provides compositions comprising reverse transcriptases
(RTs) and methods for the production of such reverse transcriptase
enzymes. Such methods provide for reverse transcriptases that are
substantially free from contamination by nucleic acids and other
unwanted materials or proteins. Compositions comprising the reverse
transcriptase enzymes of the present invention may be used in a
variety of applications, including synthesis, amplification and
sequencing of nucleic acids.
[0004] 2. Background Art and Summary of the Invention
[0005] A variety of techniques may be employed to facilitate the
preparation of intracellular proteins from microorganisms.
Typically, the initial steps in these techniques involve lysis or
rupture of the bacterial cells, to disrupt the bacterial cell wall
and allow release of the intracellular proteins into the
extracellular milieu. Following this release, the desired proteins
are purified from the extracts, typically by a series of
chromatographic steps.
[0006] Several approaches have proven useful in accomplishing the
release of intracellular proteins from bacterial cells. Included
among these are the use of chemical lysis, physical methods of
disruption, or a combination of chemical and physical approaches
(reviewed in Felix, H., Anal. Biochem. 120:211-234 (1982)).
[0007] Chemical methods of disruption of the bacterial cell wall
that have proven useful include treatment of cells with organic
solvents such as toluene (Putnam, S. L., and Koch, A. L., Anal.
Biochem. 63:350-360 (1975); Laurent, S. J., and Vannier, F. S.,
Biochimie 59:747-750 (1977); Felix, H., Anal. Biochem. 120:211-234
(1982)), with chaeotropes such as guanidine salts (Hettwer, D., and
Wang, H., Biotechnol. Bioeng. 33:886-895 (1989)), with antibiotics
such as polymyxin B (Schupp, J. M., et al., BioTechniques 19:18-20
(1995); Felix, H., Anal. Biochem. 120:211-234 (1982)), or with
enzymes such as lysozyme or lysostaphin (McHenty, C. S., and
Kornberg, A., J. Biol. Chem. 252(18):6478-6484 (1977); Cull, M.,
and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990); Hughes, A.
J., Jr., et al., J. Cell Biochem. Suppl. 016 (Part B):84 (1992);
Sambrook, J., et al., in Molecular Cloning: A Laboratory Manual,
2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press (1989), pp. 17-38; Ausubel, F. M., et al., in Current
Protocols in Molecular Biology, New York: John Wiley & Sons
(1993), pp. 4.4.1-4.47). The effects of these various chemical
agents may be enhanced by concurrently treating the bacterial cells
with detergents such as Triton X-100.RTM., sodium dodecylsulfate
(SDS) or Brij 35 (Laurent, S. J., and Vannier, F. S., Biochimie
59:747-750 (1977); Felix, H., Anal. Biochem. 120:211-234 (1982);
Hettwer, D., and Wang, H., Biotechnol. Bioeng, 33:886-895 (1989);
Cull, M., and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990);
Schupp, J. M., et al., BioTechniques 19:18-20 (1995)), or with
proteins or protamines such as bovine serum albumin or spermidine
(McHenry, C. H. and Komberg, A., J. Biol. Chem. 252(18): 6478-6484
(1977); Felix, H., Anal. Biochem. 120:211-234 (1982); Hughes, A.
J., Jr., et al., J. Cell Biochem. Suppl. 0 16 (Part B):84
(1992)).
[0008] In addition to these various chemical treatments a number of
physical methods of disruption have been used. These physical
methods include osmotic shock, e.g., suspension of the cells in a
hypotonic solution in the presence or absence of emulsifiers
(Roberts, J. D., and Lieberman, M. W., Biochemistry 18:4499-4505
(1979); Felix, H., Anal. Biochem. 120:211-234 (1982)), drying
(Mowshowitz, D. B., Anal. Biochem. 70:94-99 (1976)), bead agitation
such as ball milling (Felix, H., Anal. Biochem. 120:211-234 (1982);
Cull, M., and McHenry, C. S., Meth. Enzymol. 182:182:147-153
(1990)), temperature shock, e.g., freeze-thaw cycling (Lazzarini,
R. A., and Johnson L. D., Nature New Biol. 243:17-20 (1975); Felix,
H., Anal, Biochem. 120:211-234 (1982)), sonication (Amos, H., et
al., J. Bacteriol. 94:232-240 (1967); Ausubel, F. M., et al., in
Current Protocols in Molecular Biology, New York, John Wiley &
Sons (1993), pp. 4.4.1-4.47) and pressure disruption, e.g., use of
a french pressure cell (Ausubel, F. M., et al., in Current
Protocols in Molecular Biology, New York, John Wiley & Sons
(1993), pp. 16.8.6-16.8.8). Other approaches combine these chemical
and physical methods of disruption, such as lysozyme treatment
followed by sonication or pressure treatment, to maximize cell
disruption and protein release (Ausubel, F. M., et al., in Current
Protocols in Molecular Biology, New York, John Wiley & Sons
(1993), pp. 4.4.1-4.47).
[0009] These disruption approaches have several advantages,
including their ability to rapidly and completely (in the case of
physical methods) disrupt the bacterial cell such that the release
of intracellular proteins is maximized. In fact, these approaches
have been used in the initial steps of processes for the
purification of a variety of bacterial cytosolic enzymes, including
natural and recombinant proteins from mesophilic organisms such as
Escherichia coli, Bacillus subtilis and Staphylococcus aureus
(Laurent, S. J., and Vannier, F. S., Biochimie 59:747-750 (1977);
Cull, M., and McHenry, C. S., Meth. Enzymol. 182:147-153 (1990);
Hughes, A. J., Jr., et al., J. Cell Biochem. Suppl. 0 16 (Part
B):84 (1992); Ausubel, F. M., et al., in Current Protocols in
Molecular Biology, New York: John Wiley & Sons (1993), pp.
4.4.1-4.47), as well as phosphatases, restriction enzymes, DNA or
RNA polymerases and other proteins from thermophilic bacteria and
archaea.
[0010] However, these methods possess distinct disadvantages as
well. For example, the physical methods by definition involve
shearing and fracturing of the bacterial cell walls and plasma
membranes. These processes thus result in extracts containing large
amounts of particulate matter, such as membrane or cell wall
fragments, which must be removed from the extracts, typically by
centrifugation, prior to purification of the enzymes. This need for
centrifugation limits the batch size capable of being processed in
a single preparation to that of available centrifuge space; thus,
large production-scale preparations are impracticable if not
impossible. Furthermore, physical methods, and many chemical
techniques, typically result in the release from the cells not only
of the desired intracellular proteins, but also of undesired
nucleic acids and membrane lipids (the latter particularly
resulting when organic solvents are used). These undesirable
cellular components also complicate the subsequent processes for
purification of the desired proteins, as they increase the
viscosity of the extracts (Sambrook, J., et al., in: Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, N.Y.:
Cold Spring Harbor Laboratory Press (1989), pp 17-38; Cull, M., and
McHenry, C. S., Meth. Enzynol. 182:147-153 (1990)), and bind with
high avidity and affinity to nucleic acid-binding proteins such as
DNA polymerases, RNA polymerases and restriction enzymes.
[0011] One problem associated with these approaches is that the
enzyme preparations are typically contaminated with nucleic acids
(e.g., RNA and DNA). This contaminating nucleic acid may come not
only from the organisms which are the source of the enzyme, but
also from unknown organisms present in the reagents and materials
used to purify the enzyme after its release from the cells. Since
reverse transcriptase enzymes are routinely used in techniques of
amplification and synthesis of nucleic acid molecules (e.g., the
Polymerase Chain Reaction (PCR), particularly RT-PCR) the presence
of contaminating DNA or RNA in the enzyme preparations is a
significant problem since it can give rise to spurious
amplification or synthesis results. Thus, a need exists for
preparation of reverse transcriptase enzymes that are substantially
free of contamination by nucleic acids.
[0012] Instead of attempting to remove nucleic acids from
preparations of reverse transcriptase enzymes, a more reasonable
and successful approach would be to prevent contamination of the
enzymes by nucleic acids from the outset in the purification
process. Such an approach would be two-pronged: 1) preventing
release of nucleic acids from the bacterial cells during
permeabilization of the cells to release the enzymes; and 2)
preventing contamination of the enzymes during the purification
process itself. Furthermore, an optimal method would obviate the
need for centrifugation in the process, thus allowing large-scale,
and even continuous, production of nucleic acid-free reverse
transcriptase enzymes. The present invention provides such methods,
and reverse transcriptase enzymes produced by these methods.
[0013] The present invention generally provides methods of making a
reverse transcriptase enzyme comprising permeabilizing a cellular
source of reverse transcriptase (e.g., bacterial cells) to form
spheroplasts or protoplasts and isolating the reverse transcriptase
enzyme. Preferably, the methods are conducted under conditions
favoring the partitioning of nucleic acids from the reverse
transcriptase enzyme. In particular, the invention relates to a
method for isolation or purification of reverse transcriptases
comprising cell permeabilization, filtration and isolation.
[0014] The invention is particularly directed to methods wherein
the permeabilization of the cells is accomplished by contacting the
cells with an aqueous solution comprising at least one of: a
chaeotropic agent, preferably a guanidine salt and most preferably
guanidine hydrochloride; and/or a nonionic detergent, preferably
Triton X-100 and/or sodium deoxycholic acid. The invention is
further directed to such methods wherein the conditions favoring
the partitioning of nucleic acids from the reverse transcriptase
enzyme comprise formation of an filtrate (e.g., ultrafiltrate) by
filtration (e.g., microfiltration) of the cellular source subjected
to permeabilization (particularly of the spheroplasts or
protoplasts) through a semi-permeable membrane, which is preferably
a hydrophilic dialysis membrane, preferably in the presence of a
salt, preferably ammonium sulfate, and purification or isolation of
the reverse transcriptase enzyme from the filtrate, preferably by
chromatography using sterile materials. The invention is
particularly directed to such methods wherein bacterial cells
providing the reverse transcriptase enzyme are used, preferably
prokaryotic cells such as those of species of the genera
Escherichia (preferably E. coli), Bacillus, Serratia, Salmonella,
Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria,
Treponema, Klebsiella, Mycoplasma, Borrelia, Legionella,
Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium,
Rhizobium, Xanthomonas and Streptomyces. In another aspect, the
cellular source of reverse transcription is a recombinant cellular
source.
[0015] The invention also provides the reverse transcriptase
enzymes, or mutants, derivatives or fragments thereof, that are
made according to the methods provided. The invention is also
directed to methods for amplifying or synthesizing a nucleic acid
molecule comprising contacting a nucleic acid molecule (e.g.,
template) with an reverse transcriptase made according to the
methods of the present invention under conditions to make a first
nucleic acid molecule complementary to all or a portion of the
template. Such synthesis or amplification may further comprise
incubating the reaction with one or more polymerases (DNA
polymerases, preferably thermostable DNA polymerases such as Tne,
Tma, Taq etc. or mutants, derivatives or fragments thereof) under
conditions sufficient to make a second nucleic acid molecule
complementary to all or a portion of the first nucleic acid
molecule.
[0016] The invention also provides kits for amplifying or
synthesizing nucleic acid molecules comprising a carrier means
having in close confinement therein one or more container means,
wherein said kit may comprise at least one component selected from
one or more reverse transcriptases produced according to the
invention, one or more polymerases (e.g., DNA polymerases), one or
more nucleotides or derivatives thereof, one or more primers, and
one or more synthesis or amplification reaction buffers.
[0017] Other features and advantages of the present invention will
be apparent to those skilled in the art from the following
description of the preferred embodiments and from the claims.
DETAILED DESCRIPTION OF THE INVENTION
Overview
[0018] The present invention in a preferred aspect provides reverse
transcription enzymes that are substantially pure and more
preferably reverse transcriptases that are substantially free of
nucleic acids. As used herein, the term "substantially free of
nucleic acids" means an enzyme composition that comprises no
nucleic acids, or that comprises nucleic acids below the level of
detection, when assayed by standard biochemical assays for nucleic
acids. Such assays may include gel electrophoresis (e.g., agarose
gel electrophoresis coupled with nucleic acid staining such as
ethidium bromide, acridine orange or Hoechst staining),
spectrophotometry (e.g., ultraviolet, atomic absorption, NMR or
mass spectrometry), chromatography (liquid, gas, HPLC or FPLC), or
by functional assays for nucleic acids detection such as
amplification. An example of such functional assay is based on
measuring incorporation of labeled nucleotides (e.g., radio
labeled, enzyme labels, chemiluminescent labels, etc.) by the
enzyme preparation in a "no-template" nucleic acid amplification
reaction. These biochemical and functional assays are described in
more detail below and in Example 3. The invention also provides
methods for the production of these enzymes, and compositions and
kits comprising these enzymes which may be used in synthesis or
amplifying nucleic acid molecules, including through use of the
polymerase chain reaction (PCR).
[0019] Briefly summarized, the present invention utilizes a scheme
comprising permeabilization of cells (preferably bacterial cells)
to form spheroplasts or protoplasts, filtration (e.g.,
microfiltration) of the spheroplasts or protoplasts to form a
filtrate (e.g., microfiltrate), ultrafiltration of the filtrate to
form an ultrafiltrate, and purification of the enzyme from the
ultrafiltrate, preferably by conventional liquid chromatography.
The present invention 1) provides a method of large-scale (>20
million unit) production of reverse transcriptases, including
MMLV-RT and mutants or derivatives thereof; and 2) provides a
scalable method for the production of any desirable quantity of the
desired enzyme.
[0020] The present methods are based in particular upon an
optimized method of chemical permeabilization of cells (preferably
bacterial cells), which preferably strips the cell wall and yields
spheroplasts (although conditions to merely permeabilize the cell
wall to form protoplasts may equally be used), and an optimized
method of filtration of the spheroplasts or protoplasts under
conditions favoring the release of reverse transcriptase enzymes,
but inhibiting the release of nucleic acids, from the spheroplasts
or protoplasts. The permeabilization process has been optimized to
allow intracellular enzymes, particularly reverse transcriptases,
to permeate or cross the spheroplast or protoplast membrane while
preventing the cellular nucleic acids (DNA and/or RNA) from
entering the permeation buffer. This approach provides an initial
extract that is enriched in enzyme and that is relatively free of
nucleic acids. The extract is then subjected to filtration under
conditions (including precise definition of the variables of salt,
pH, and choice of membrane chemistry) favoring release of the
enzyme from the spheroplasts or protoplasts while preventing cells,
cell debris and/or nucleic acids from crossing the filtration
membrane barriers. Following filtration (which may include
microfiltration and/or ultrafiltration), reverse transcriptase
enzymes may be purified or isolated by standard techniques such as
chromatography or electrophoresis, to provide enzyme preparations
of the invention.
Sources of Reverse Transcriptase Enzymes
[0021] Any reverse transcriptase enzymes may be prepared according
to the methods of the present invention from a variety of
prokaryotic and eukaryotic cells including bacteria that are
commercially available (for example, from American Type Culture
Collection (ATCC), Rockville, Md. and the Collection, Agricultural
Research Culture Collection (NRRL), Peoria, Ill.). Examples of
bacterial deposits as sources of RTs include ATCC deposit no. 67007
(M-MLV RT 11+), ATCC deposit no. 67555 (M-MLV H-), NRRL B-21790
(AMV RT .alpha.H.sup.+/.beta.H.sup.-), and NRRL B-21679 (RSV RT
.alpha.H.sup.+/.beta.H.sup.-).
[0022] Enzymes prepared in accordance with the invention include
any enzyme having reverse transcriptase activity. Such enzymes
include, but are not limited to, retroviral reverse transcriptase,
retrotransposon reverse transcriptase, hepatitis B reverse
transcriptase, cauliflower mosaic virus reverse transcriptase,
bacterial reverse transcriptase, and mutants, fragments, variants
or derivatives thereof (see WO 98/47912, U.S. Pat. Nos. 5,668,005,
and 5,017,492). As will be understood by one of ordinary skill in
the art, modified reverse transcriptases may be obtained by
recombinant or genetic engineering techniques that are routine and
well-known in the art. Mutant reverse transcriptases can, for
example, be obtained by mutating the gene or genes encoding the
reverse transcriptase of interest by site-directed or random
mutagenesis. Such mutations may include point mutations, deletion
mutations and insertional mutations. Preferably, one or more point
mutations (e.g., substitution of one or more amino acids with one
or more different amino acids) are used to construct mutant reverse
transcriptases of the invention. Fragments of reverse
transcriptases may be obtained by deletion mutation by recombinant
techniques that are routine and well-known in the art, or by
enzymatic digestion of the reverse transcriptase(s) of interest
using any of a number of well-known proteolytic enzymes.
[0023] Preferred enzymes which may be prepared according to the
invention include those that are reduced or substantially reduced
in RNase H activity. Such enzymes that are reduced or substantially
reduced in RNase H activity may be obtained by mutating the RNase H
domain within the reverse transcriptase of interest, preferably by
one or more point mutations, one or more deletion mutations, and/or
one or more insertion mutations as described above. By an enzyme
"substantially reduced in RNase H activity" is meant that the
enzyme has less than about 30%, less than about 25%, less than
about 20%, more preferably less than about 15%, less than about
10%, less than about 7.5%, or less than about 5%, and most
preferably less than about 5% or less than about 2%, of the RNase H
activity of the corresponding wild type or RNase H+ enzyme such as
wild type Moloney Murine Leukemia Virus (M-MLV), Avian
Myeloblastosis Virus (AMV) or Rous Sarcoma Virus (RSV) reverse
transcriptases. The RNase H activity of any enzyme may be
determined by a variety of assays, such as those described, for
sample, in U.S. Pat. No, 5,244,797, in Kotewicz, M. L., et al.,
Nucl. Acids Res. 16:265 (1988), in Gerard, G. F., et al., FOCUS
14(5):91 (1992), in WO 98/47912, and in U.S. Pat. No. 5,668,005,
the disclosures of all of which are fully incorporated herein by
reference.
[0024] Particularly preferred enzymes for use in the invention
include, but are not limited to M-MLV H-reverse transcriptase, RSV
H-reverse transcriptase, AMV H-reverse transcriptase, RAV H-reverse
transcriptase, MAV reverse transcriptase and HIV H-reverse
transcriptase (see WO 98/47912). It will be understood by one of
ordinary skill, however, that any enzyme capable of producing a DNA
molecule from a ribonucleic acid molecule (i.e., having reverse
transcriptase activity) that is reduced or not reduced in RNase H
activity may be equivalently prepared in accordance with the
invention.
[0025] It will be understood by one of ordinary skill in the art,
however, that any cell, virus, microorganism or bacteria (including
prokaryotic and eukaryotic) may be used as a source for preparation
of reverse transcriptase enzymes (e.g., cellular source of RT)
according to the methods of the present invention. Preferably,
recombinant cells (prokaryotic or eukaryotic) are used as a source
of the reverse transcriptases in the methods of the invention. Such
recombinant cells may be prepared by recombinant DNA techniques
that are familiar to one or ordinary skill in the art (see e.g.,
Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis, D.
A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372-3376
(1988)). Such sources of reverse transcriptases may be grown
according to standard microbiological techniques, using culture
media and incubation conditions suitable for growing active
cultures of the particular species that are well-known to one of
ordinary skill in the art (see, e.g., Brock, T. D., and Freeze, H.,
J. Bacteriol. 98(1):289-297 (1969); Oshima, T., and Imahori, K.,
Int. J. Syst. Bacteriol. 24(1):102-112 (1974)).
Permeabilization of Cells
[0026] In the initial steps of the present methods, a cellular
source of reverse transcriptase is treated under conditions to
allow the release of the reverse transcriptase from the cell and
preferably to retain nucleic acids in the cell. Such conditions may
include permeabilizing the cells by stripping away the cell walls
and converting the cells into spheroplasts or permeabilizing the
cell (making openings in the cell wall without totally removing it)
to convert the cells into protoplasts. Such conditions may include
chemical and/or enzymatic (e.g., lysozyme) treatment, although a
variety of other techniques may be used for this permeabilization.
The production of substantially nucleic acid-free enzymes by the
present invention preferably uses a permeabilization method which
will produce protoplast or spheroplasts that retain substantially
all the nucleic acids within the spheroplast or protoplasts while
allowing intracellular proteins (including enzymes) to move across
the spheroplast or protoplast membrane. All procedures from
permeabilization to final purification of the enzymes should be
carried out at temperatures below normal room temperature,
preferably at about 1-10.degree. C., more preferably at about
2-8.degree. C., and most preferably at about 2-6.degree. C., to
prevent enzyme denaturation and loss of activity. Furthermore, all
materials used throughout the present methods (i.e., reagents,
salts, chromatography resins, equipment) should be sterilized by
heat or barrier sterilization techniques (as appropriate to the
material to be sterilized), to prevent the contamination of the
reverse transcriptase enzymes with nucleic acids or other unwanted
contaminants.
[0027] This permeabilization is preferably accomplished by
suspension of the cells in an aqueous solution comprising at least
one or more chaeotropic agents and/or nonionic detergent. According
to a preferred embodiment, this permeabilization is preferably
accomplished by suspension of the cells in an aqueous solution
comprising at least two nonionic detergents. Chaeotropic agents
preferable for use in the methods of the present invention include
salts of guanidine or urea, most preferably guanidine
hydrochloride. Any nonionic detergent may be used; most preferable
are octylphenoxy-polyethoxyethanol nonionic surfactant (TRITON
X-100.RTM.), Brij 35, Tween 20 and Nonidet P-40 (NP-40.RTM.),
although other nonionic surfactants and mixtures thereof, such as
N-alkylglucosides, N-alkylmaltosides, glucamides, digitonin,
deoxycholate,
3-[3-cholamidopropyl)dimethyl-ammonium]-1-propane-sulfonate (CHAPS)
or cetyltrimethyl-ammonium-bromide (CTAB) may also be used in the
present compositions. Reagents such as chaeotropes, detergents,
buffer salts, etc., are available commercially, for example from
Sigma Chemical Co. (St. Louis, Mo.).
[0028] For permeabilization, the cells are preferably suspended in
a buffered salt solution containing the chaeotrope(s) and/or the
detergent(s). Preferably, the solution is an aqueous solution with
a distilled, deionized water (dH.sup.2O) base consisting of
bis-trishydroxymethylaminomethane (BisTRIS.RTM. base) at a
concentration of about 25-500 mM, preferably about 50-250 mM, more
preferably about 50-150 mM, and most preferably about 100 mM, at a
pH of about 7.0-9.0, preferably about 7.0-8.5, more preferably
about 7.0-8.0, more preferably about 7.0-7.5 and most preferably
about 7.0 (pH at about 20-25.degree. C.). The concentration of the
chaeotrope in the solution is preferably about 300-1000 mM, more
preferably about 500-750 mM, and most preferably about 600 mM. The
concentration of the nonionic detergent is preferably about 1-10%
(vol/vol), more preferably about 2-8% and most preferably about
2-5%. Within the context of the present invention, one or more
chaeotropic agents and/or nonionic detergents may be used within
the concentration ranges specified. The permeabilization buffer
solution may also comprise other components, such as protease
inhibitors (e.g., phenylmethylsulfonylfluoride, added at a final
concentration of about 0.5 mM), reducing agents (e.g.,
.beta.-mercaptoethanol or most preferably dithiothreitol at a final
concentration of about 1 mM),and chelating agents (e.g., disodium
ethylenediaminetetraacetic acid (Na.sub.2EDTA), most preferably at
a concentration of about 10 mM); this buffer composition is
referred to hereinafter as "permeabilization buffer." It will be
understood by one of ordinary skill in the art, however, that other
suitable buffer compositions may be substituted with equivalent
effect in the permeabilization process.
[0029] For permeabilization, the cells are preferably suspended in
permeabilization buffer at a concentration of about 50-1000 g (wet
weight) of cells per liter of solution, preferably about 100-500
g/L, and most preferably about 250 g/L (cell density of about
1-5.times.10.sup.10 cells/gram, preferably about
2-5.times.10.sup.10 cells/gram, and most preferably about
2.5.times.10.sup.10 cells/gram). The cell suspension is gently
stirred, preferably via magnetic or impeller stirring, in such a
way as to prevent shearing and rupture of the cells. After about
30-60 minutes, most preferably about 45 minutes, a
protein-extracting salt is added to the suspension to enhance the
permeation of the intracellular enzymes across the spheroplast or
protoplast membranes. Although any salt may be used in the present
invention (except salts of toxic metals such as cadmium or other
heavy metals), preferred salts include sodium chloride, potassium
acetate, sodium acetate, ammonium acetate, ammonium chloride,
ammonium sulfate or potassium chloride, most preferably ammonium
sulfate. Salt is added to the suspension at a concentration of
about 100-500 mM, preferably about 200-400 mM, and most preferably
about 300 mM. The salt should be gradually added to the solution to
provide for optimal solubilization. Following addition of the salt,
the solution is mixed for about an additional 30-60 minutes, most
preferably about an additional 45 minutes, during which time the
bacterial cells are converted into spheroplasts or protoplast and
the intracellular proteins, including reverse transcriptase
enzymes, begin to cross the spheroplast or protoplast membrane
while cellular nucleic acids are preferably retained within the
spheroplast or protoplast. Microfiltration, Concentration and
Diafiltration
[0030] Following permeabilization of the cells, reverse
transcriptases are collected by subjecting the spheroplasts or
protoplast to filtration (e.g., microfiltraiton) to separate the
enzymes from the spheroplasts or protoplast and remove particulate
matter. In another aspect, the filtrate may be subjected to
concentration and/or diafiltration. The present methods obviates
the need for precipitation of nucleic acids and/or the use of
centrifugation techniques; this elimation of centrifugation
facilitates the rapid production of reverse transcriptase enzymes
at any scale in a continuous or discontinuous fashion. The general
methods of filtration (e.g., microfiltration), concentration and
diafiltration are generally well-known to one of ordinary skills,
and will result in the preparation of an enzyme ultrafiltrate
(which is preferably nucleic-acid free) suitable for purification
and characterization of the enzymes.
[0031] Microfiltration is preferably carried out by collecting the
spheroplast/or protoplast solution in permeabilization buffer
(described above) and diafiltering the solution against a
filtration buffer thorugh a semi-permeable membrane, most
preferably a hydrophilic dialysis, microfiltration or
ultrafiltration membrane. The filtration buffer preferably is a
dH2O-based soltion comprising: a) a buffer salt, preferably
trishydroxymethylaminomethane (TRIS base) at a concentration of
about 25-500 mM, preferably about 50-250 mM, more preferably about
50-150 mM, and most preferably about 100 mM, at a pH of about
7.0-9.0, preferably about 7.0-8.5, more preferably about 7.0-8.0,
and most preferably about 8.0 (pH at 4.degree. C.); and b) the
protein-extracting salt which was added to the permeabilization
buffer, which is preferably ammonium sulfate, at a concentration of
about 100-500 mM, preferably about 200-400 mM, and most preferably
about 300 mM. The filtration buffer solution may also comprise
other components, such as protease inhibitors (e.g.,
phenylmethysulfonylfluoride, added at a final concentration of
about 1.0 mM), reducing agents (e.g., .beta.-mercaptoethanol or
most preferably dithiothreitol at a final concentration of about 1
mM), and chelating agents (e.g., disodium
ethylenediaminetetraacetic acid (Na.sub.2EDTA), most preferably at
a concentration of about 10 mM; this buffer composition is referred
to hereinafter as "filtration buffer." It will be understood by one
of ordinary skill in the art, however, that other suitable buffer
compositions may be substituted with equivalent effect in the
filtration process.
[0032] Preferable for use in microfiltration is a system allowing
permeation of intracellular enzymes through the membrane and into
the filtrate, leaving spheroplasts and/or protoplast (with the
nucleic acids retained therein) and particulate matter in the
retentate. One suitable system providing such conditions is, for
example, a hollow fiber microfiltration system which is
commercially available (Spectrum), although similar systems
providing the same results will be known to one of ordinary skill.
Following microfiltration in this manner, the filtrate contains the
reverse transcriptase enzymes which are substantially free of
nucleic acids such as DNA, as the DNA is partitioned from the
enzymes by being retained with the particulate matter. This
filtrate may then be concentrated, for example by membrane
concentration through a semi-permeable membrane using a
commercially available system (AG/Technology Corp.) or equivalent.
The enzymes may then be individually purified from the concentrate
as described below; alternatively, the concentrate may be
diafiltered as described above against a suitable buffer solution
to place the enzymes into an appropriate chemical environment for
purification, as described in more detail in Example 2.
Purification and Characterization of Enzymes
[0033] Following concentration and/or diafiltration as described
above, reverse transcriptase enzymes may be purified by a variety
of protein purification techniques that are well-known to one of
ordinary skill in the art. Suitable techniques for purification
include, but are not limited, ammonium sulfate or ethanol
precipitation, acid extraction, preparative gel electrophoresis,
immunoadsorption, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, immunoaffinity
chromatography, size exclusion chromatography, liquid
chromatography (LC), high performance LC (HPLC), fast performance
LC (FPLC), hydroxylapatite chromatography, lectin chromatography,
and immobilized metal affinity chromatography (IMAC). Most
preferably, the enzymes are purified by a combination of liquid
chromatographic techniques including ion exchange, affinity and
size exclusion methods such as those described in Example 3,
although alternative chromatographic solid supports, mobile phases
and associated methods may be equivalently used and will be
well-known to one of ordinary skill. The invention thus provides
for substantially pure reverse transcriptases. Substantially pure a
used herein refers to a preparation or sample which is
substantially free of contaminating components, proteins etc. which
may adversely affect the activity or performance of the RT in the
use of the enzyme such as in amplification or synthesis.
Assays for Nucleic Acid Content
[0034] Purified reverse transcriptase enzymes made according to the
present invention may be examined for nucleic acid content by a
variety of methods which are well-known to one of ordinary skill in
the art. For example, a sample of the final product can be assayed
by ultraviolet spectrophotometry, comparing absorption of light by
the sample at a wavelength of 260 nm (A.sub.260, the absorption
maximum for DNA) to that at 280 nm (A.sub.280, the absorption
maximum for tryptophan, which is found in most proteins); the lower
the A.sub.260/A.sub.280 ratio, the lower the content of DNA in the
sample. Samples with minimal A.sub.260/A.sub.280 values may then be
pooled to constitute a substantially nucleic acid-free preparation
of reverse transcriptase enzymes.
[0035] Alternatively, samples may be directly assayed for the
presence of DNA or other nucleic acids by gel electrophoresis or
dot blotting and staining with a DNA-binding dye (e.g., ethidium
bromide, acridine orange, Hoechst stain, pico green) or antibody,
which are commercially available, for example, from Sigma (St.
Louis, Mo.). In addition, the DNA content of samples of reverse
transcriptases may be examined by carrying out an amplification
reaction in the absence of exogenously added DNA template, either
as a "no-template control" in a standard PCR assay (Rand, K. H.,
and Houck, H., Mol. Cell Probes 4(6):445-450 (1990)), or by
specifically designing an assay to measure DNA content by
radiolabeled nucleotide incorporation into salmon testes or bovine
thymus DNA, according to methods that are standard in the art. Use
of such assays will allow one of ordinary skill, without undue
experimentation, to identify samples of reverse transcriptase
enzymes obtained by the purification schemes described above, which
may then be pooled and used as preparations of substantially
nucleic acid-free reverse transcriptase enzymes.
Formulation of Enzymes
[0036] Following their purification or isolation, the substantially
pure and preferably substantially DNA-free reverse transcriptase
enzymes may be stored until use in a buffered solution at
temperatures of about -.phi.80.degree. to 25.degree. C., most
preferably at -80.degree. to 4.degree. C., or in lyophilized form.
Alternately, the enzymes may be stabilized by drying in the
presence of a sugar such as trehalose (U.S. Pat. Nos. 5,098,893 and
4,824,938) or acacia gum, pectin, carboxymethylcellulose,
carboxymethyl-hydroxyethylcellulose, guar, carboxy guar,
carboxymethylhydroxypropyl guar, laminaran, chitin, alginates or
carrageenan. In addition, the enzymes provided by the present
invention may be directly formulated into compositions to be used
in techniques requiring the use of reverse transcriptase enzymes,
such as compositions for nucleic acid synthesis or
amplification.
Kits
[0037] In other preferred embodiments, the substantially pure and
preferably substantially DNA-free reverse transcriptases provided
by the present invention may be assembled into kits for use in
methods requiring reverse transcriptase enzymes, such as nucleic
acid synthesis (e.g., cDNA synthesis), amplification (e.g., RT-PCR)
or sequencing utilizing RT. The kit according to the present
invention comprises a carrier means having in close confinement
therein one or more container means, such as vials, tubes, bottles
and the like, wherein a first container means contains a reverse
transcriptase of this invention. The kit encompassed by this aspect
of the present invention may further comprise in the same or
different containers additional reagents and compounds necessary
for carrying out standard nucleic synthesis, amplification and
sequencing protocols. Such additional components may include
reaction buffers, nucleotides (e.g., dTTP, dATP, dCTP, dGTP, ddATP,
ddTTP, ddGTP, ddCTP and derivatives thereof including labeled
nucleotides), one or more DNA polymerases (such as Taq DNA
polymerase), one or more primers and the like.
Use of the Reverse Transcriptase Enzymes
[0038] The substantially pure or substantially DNA-free reverse
transcriptase enzymes and kits embodied in the present invention
will have general utility in any application utilizing reverse
transcriptase enzymes, including but not limited to nucleic acid
cDNA synthesis, and nucleic acid amplification or sequencing
methodologies.
[0039] In a first aspect, the RTs of the invention may be used for
synthesis of nucleic acid molecules. Such methods for making one or
more nucleic acid molecules, comprising mixing one or more nucleic
acid templates (preferably one or more RNA templates and most
preferably one or more messenger RNA templates) with one or more
polypeptides having reverse transcriptase activity and incubating
the mixture under conditions sufficient to make a first nucleic
acid molecule or molecules complementary to all or a portion of the
one or more nucleic acid templates. In a preferred embodiment, the
first nucleic acid molecule is a single-stranded cDNA. Nucleic acid
templates suitable for reverse transcription according to this
aspect of the invention include any nucleic acid molecule or
population of nucleic acid molecules (preferably RNA and most
preferably mRNA), particularly those derived from a cell or tissue.
In a preferred aspect, a population of mRNA molecules (a number of
different mRNA molecules, typically obtained from cells or tissue)
are used to make a cDNA library, in accordance with the invention.
Preferred cellular sources of nucleic acid templates include
bacterial cells, fungal cells, plant cells and animal cells.
[0040] RT enzymes made in accordance with the invention may also be
used in methods for amplifying and sequencing nucleic acid
molecules. Nucleic acid amplification methods according to this
aspect of the invention may be one step (e.g., one-step RT-PCR) or
two-step (e.g., two-step RT-PCR) reactions. According to the
invention, one-step RT-PCR type reactions may be accomplished in
one tube thereby lowering the possibility of contamination. Such
one-step reaction comprise (a) mixing a nucleic acid template
(e.g., mRNA) with one or more polypeptides having reverse
transcriptase activity and with one or more DNA polymerases and (b)
incubating the mixture under conditions sufficient to amplify a
nucleic acid molecule complementary to all or a portion of the
template with one or more polypeptides having reverse transcriptase
activity (and optionally having DNA polymerase activity).
Incubating such a reaction mixture under appropriate conditions
allows amplification of a nucleic acid molecule complementary to
all or a portion of the template. Such amplification may be
accomplished by the reverse transcriptase activity alone or in
combination with the DNA polymerase activity. Two-step RT-PCR
reactions may be accomplished in two separate steps. Such a method
comprises (a) mixing a nucleic acid template (e.g., mRNA) with one
or more reverse transcriptases, (b) incubating the mixture under
conditions sufficient to make a nucleic acid molecule (e.g., a DNA
molecule) complementary to all or a portion of the template, (c)
mixing the nucleic acid molecule with one or more DNA polymerases
and (d) incubating the mixture of step (c) under conditions
sufficient to amplify the nucleic acid molecule. For amplification
of long nucleic acid molecules (i.e., greater than about 3-5 Kb in
length), a combination of DNA polymerases may be used, such as one
DNA polymerase having 3' exonuclease activity and another DNA
polymerase being substantially reduced in 3' exonuclease activity.
An alternative two-step procedure comprises the use of one or more
polypeptides having reverse transcriptase activity and DNA
polymerase activity (e.g., Tth, Tma or Tne DNA polymerases and the
like) rather than separate addition of a reverse transcriptase and
a DNA polymerase.
[0041] Nucleic acid sequencing methods according to this aspect of
the invention may comprise both cycle sequencing (sequencing in
combination with amplification) and standard sequencing reactions.
The sequencing method of the invention thus comprises (a) mixing a
nucleic acid molecule to be sequenced with one or more primers, two
or more reverse transcriptases, one or more nucleotides and one or
more terminating agents, (b) incubating the mixture under
conditions sufficient to synthesize a population of nucleic acid
molecules complementary to all or a portion of the molecule to be
sequenced, and (c) separating the population to determine the
nucleotide sequence of all or a portion of the molecule to be
sequenced. According to the invention, one or more DNA polymerases
(preferably thermostable DNA polymerases) may be used in
combination with or separate from the reverse transcriptases.
[0042] Amplification methods in which the present enzymes may be
used include PCR (U.S. Pat. Nos. 4,683,195 and 4,683,202), Strand
Displacement Amplification (SDA; U.S. Pat. No. 5,455,166; EP 0 684
315), and Nucleic Acid Sequence-Based Amplification (NASBA; U.S.
Pat. No. 5,409,818; EP 0 329 822). Nucleic acid sequencing
techniques which may employ the present enzymes include dideoxy
sequencing methods such as those disclosed in U.S. Pat. Nos.
4,962,022 and 5,498,523, as well as more complex PCR-based nucleic
acid fingerprinting techniques such as Random Amplified Polymorphic
DNA (RAPD) analysis (Williams, J. G. K., et al., Nucl. Acids Res.
18(22):6531-6535, 1990), Arbitrarily Primed PCR (AP-PCR; Welsh, J.,
and McClelland, M., Nucl. Acids Res. 18(24):7213-7218, 1990), DNA
Amplification Fingerprinting (DAF; Caetano-Anolles et al.,
Bio/Technology 9:553-557 (1991)) microsatellite PCR or Directed
Amplification of Minisatellite-region DNA (DAMD; Heath, D. D., et
al., Nucl. Acids Res. 21(24):5782-5785 (1993)), and Amplification
Fragment Length Polymorphism (AFLP) analysis (EP 0 534 858; Vos,
P., et al., Nucl. Acids Res. 23(21):4407-4414 (1995); Lin, J. J.,
and Kuo, J., FOCUS 17(2):66-70 (1995)). In particular, the enzymes
and kits of the present invention will be useful in the fields of
medical therapeutics and diagnostics, forensics, and agricultural
and other biological sciences, in any procedure utilizing reverse
transcriptase enzymes.
[0043] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLES
Example 1
Permeabilization of Bacterial Cells
[0044] In the initial steps of the purification process, 20 kg
bacterial cells (E.coli, N4830 (pRT601) (see U.S. Pat. No.
5,017,492; ATCC deposit no. 67007) containing the expression vector
for MMLV-RT which were obtained directly from actively growing
cultures were suspended at 250 g of cells/L into cold (4.degree.
C.) permeabilization buffer (100 mM BisTRIS, 5.0% Triton X-100,
2.0% sodium deoxycholic acid, 10 mM EDTA, 1 mM dithiothreitol
(DTT), pH 7.0.
[0045] During suspension of the cells in the buffer,
phenylmethylsulfonylfluoride (PMSF) was added to a final
concentration of 1.0 mM. Cells were stirred for about 45 minutes at
4.degree. C. to ensure complete suspension, and then ammonium
sulfate was added to a final concentration of 300 mM and the cell
suspension was stirred for an additional 45 minutes. During this
time, cells were permeabilized via the action of the deoxycholic
acid and Triton X-100, and intracellular protein release into the
buffer was enhanced by the action of the ammonium sulfate.
Example 2
Microfiltration, Concentration and Diafiltration of Extracts
[0046] Microfiltration of the suspension was then carried out
through 120 ft.sup.2 0.2 .mu.m Microgon mixed ester cellulose
hollow fiber system, using a re-circulation rate of 120 L/min. The
suspension was diafiltered with five to six volumes of cold
filtration buffer, collecting the permeate in a suitable sized
chilled (4.degree. C.) container. Under these conditions,
recombinant enzymes passed through the membrane with the permeate,
leaving the bacterial cells in the retentate.
[0047] As the ultrafiltration proceeded, concentration of the
permeate was begun once a sufficient volume had been collected to
prime the second ultrafiltration system. Permeate was concentrated
using an Amicon DC-90 system, through an AG technologies 10,000
MWCO membrane (although alternative membrane systems of 10,000
MWCO, such as a Filtron system, a Millipore plate and frame system,
or a membrane from Microgon may be also used) and an in-line
chiller to minimize heat build-up from the pumping system. Permeate
was concentrated to approximately the original volume of the
extract (see Example 1), and was then diafiltered against about
seven volumes of diafiltration buffer (20 mM NaPi, 100 mM NaCl,
10.0 mM EDTA, 1 mM DTT, pH 6.5), until the conductivity was <7
mS. Ultrafiltrate was then immediately used for purification of the
enzyme (Example 3).
Example 3
Purification and Characterization of DNA-free Enzyme
[0048] Purification of the enzyme from the ultrafiltrate was
accomplished by a series of chromatographic steps, using a
procedure modified slightly from that described for purification of
T5 DNA polymerase from E. coli (Hughes, A. J., Jr., et al., J. Cell
Biochem. Suppl. 0 16(Part B):84 (1992)).
A. Macroprep High S
[0049] The filtrate was mixed with 9L Whatman DE-52 and then was
polish filtered through two CUNO 8ZP 10 A depth filters. In the
first chromatographic step, the ultrafiltrate was applied to a 9L
BioRad Macroprep High S. The column was then washed with 10 volumes
of 20 mM TRIS, 150 mM NaCl, 0.1 mM EDTA, 10% glycerol, 0.01% Triton
X-100, 1 mM DTT, pH 8.0 at 4.0.degree. C. run at a flow rate of
about 20 cm/hr. Product elution was effected with a ten column
volume gradient of the wash buffer to this same buffer containing
800 mM NaCl w/o EDTA run at 10 cm/hr. Fractions demonstrating at
least 1/3 of the large UV peak were pooled and subjected to further
purification.
B. Macroprep Ceramic HTP
[0050] Pooled eluate from the High S Column was applied at a
flow-rate of 20 cm/hr to a 6L column of Macroprep Ceramic HTP, and
the column was then washed with 5 volumes of 20 mM potassium
phosphate, 100 mM KCl, 10% glycerol, 0.01% Triton X-100, 1 mM DTT,
pH 7.0 at 4.degree. C. at a flow-rate of 10 cm/hr. Fractions
showing at least 1/5 of the UV peak were pooled and subjected to
further purification.
C. Fractogel COO-Column
[0051] The pool from the Ceramic HTP column was diluted with an
equal volume of 100 mM TRIS, 100 mM NaCl, 0.2 mM EDTA, 30%
glycerol, 0.01% Triton X-100, 1 mM DTT, pH 7.5 at 4.0.degree. C.
and applied at a flow rate of 20 cm/hr to a Fractogel COO-column
(E. Merck, Inc.), which concentrates the product. The column was
then washed with 2 column volumes of low salt Fractogel COO-buffer
(20 mM TRIS, 100 mM NaCl, 20% glycerol, 0.1 mM EDTA, 0.01% Triton
X-100, 1 mM DTT, pH 7.5), and the enzyme eluted with 50% high salt
ceramic Fractogel COO-buffer (20 mM TRIS, 400 mM NaCl, 0.1 mM EDTA,
20% glycerol, 0.01% Triton X-100, 1.0 mM DTT, pH 7.5 at 4.0.degree.
C. at 20 cm/hr, and fractions containing the UV peak were
collected, and pooled as described above.
D. Dialysis
[0052] Fractogel COO-pool was dialyzed against 20 volumes of
dialysis buffer (20 mM TRIS, 0.1 mM EDTA, 50% (vol/vol) glycerol,
100 mM NaCl, 0.01% Triton X-100, 1 mM DTT, pH 7.5) for 24 hours.
Purified enzyme bulk was then stored at -20.degree. C. until
used.
E. DNA Contamination Assays
[0053] To determine the extent of DNA contamination of various
preparations of RT, samples of RT obtained from commercial sources
may be compared to a preparation made according to the methods of
the present invention in a polymerase assay similar to that
outlined above, except that no salmon testes DNA template is
included in the reaction mixture. Briefly, reaction mixtures (500
.mu.l) containing 25 mM TAPS (pH 9.3), 10 mM MgCl.sub.2, 50 mM KCl,
1 mM DTT, 100 .mu.M each of dATP, dTTP, dGTP and dCTP, and 600 cpm
of [.sup.3H]dTTP/pmol of total nucleotide are prepared and
pre-incubated at 72.degree. C. for five minutes. 100 units of M-MLV
RT are added to the reaction mixtures and then 100 units of
purified DNA-free Taq DNA polymerase (see U.S. Pat. No. 5,861,295)
are added at specific time intervals to initiate the reaction. A 30
.mu.l sample is removed and added to a vial containing 5 .mu.l of
500 mM EDTA on ice. Once all time points are collected, a 20 .mu.l
aliquot of the quenched reaction sample is applied to a GF/C
filter, which is washed, dried and counted as described above.
Results are expressed as 3H incorporation (cpm) at each time
point.
[0054] Other commercially available preparations of RT may be
compared to the preparations provided by the present invention for
their DNA content. Together, the results should indicate that
preparations of RT provided by the present invention are
substantially free of nucleic acids, while several commonly used
commercial preparations of RT contain substantial amounts of
contaminating DNA.
[0055] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be understood by those of ordinary skill
in the art that the same can be performed by modifying or changing
the invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0056] All publications, patents and patent applications cited
herein are indicative of the level of skill of those skilled in the
art to which this invention pertains, and are herein incorporated
by reference in their entirety.
* * * * *