U.S. patent number RE35,443 [Application Number 08/292,574] was granted by the patent office on 1997-02-04 for purification of q beta replicase.
This patent grant is currently assigned to Amoco Corporation. Invention is credited to Karin Borcherts Mahdavie, Robert A. DiFrancesco.
United States Patent |
RE35,443 |
DiFrancesco , et
al. |
February 4, 1997 |
Purification of Q Beta replicase
Abstract
A process of producing highly pure, Q Beta replicase having a
high level of activity is described. The present process allows
isolation of Q Beta replicase from recombinant bacteria containing
a clone of a phage DNA encoding the 65,000 weight subunit of Q Beta
replicase. The present process provides an efficient method for
producing pure Q Beta replicase which can readily be scaled up to
commercial production levels.
Inventors: |
DiFrancesco; Robert A.
(Flemington, NJ), Borcherts Mahdavie; Karin (Newton,
MA) |
Assignee: |
Amoco Corporation (Chicago,
IL)
|
Family
ID: |
23433925 |
Appl.
No.: |
08/292,574 |
Filed: |
August 18, 1994 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
364306 |
Jun 9, 1989 |
05141857 |
Aug 25, 1992 |
|
|
Current U.S.
Class: |
435/194;
424/94.5; 435/220; 435/849 |
Current CPC
Class: |
C12N
9/127 (20130101) |
Current International
Class: |
C12N
9/12 (20060101); C12N 009/12 () |
Field of
Search: |
;435/194,849,91,220
;424/94.61 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4693978 |
September 1987 |
Stetter et al. |
4786600 |
November 1988 |
Kramer et al. |
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Other References
K Kobayashi, Patent Abstracts of Japan, vol. 8, No. 77, C-218,
(abstract JP 57-111364 (1982). .
D. Hill and T. Blumental, Nature, 301:305-352 (1983). .
Hill and Blumenthal, Nature 301:350-352 (1983). .
Banerjee et al., J. Mol. Biol. 45:181-193 (1986). .
M. Sumper and R. Luce, Proc. Nat'l. Acad. Sci. USA, 72:162-166
(1975). .
R. Kamen, Biochimica and Biophysica Acta, 262:88-100 (1972). .
J. Yamauchi, Patent Abstracts of Japan, vol. 10, No. 23, C-325
(abstract of JP 60-176584) (1984). .
C. K. Biebricher et al., Nature, 321:89-91 (1986). .
Schattner et al., J. Mol. Biol. 117:877-907 (1977). .
Lizardi et al Bio/technology (vol. 6) pp. 1197-1201 1988. .
K. Kobayashi, Patent Abstracts of Japan, vol. 7, No. 273, C-198
(abstract JP 57-33969) (1982). .
T. Blumenthal and G. G. Carmichael, Annual Review of Biochemistry,
48:525-548 (1979). .
D. R. Mills, Journal of Molecular Biology, 200:489-500. .
D. R. Mills et al., Science, 180:916-927 (1973). .
N. V. Fedoroff, In: Dissertation Abstracts Int'l., Sci. and Eng.,
34(1):110B-111B (1973). .
Cooper, "The Tools of Biochemistry" ch. 4, pp. 136-151 1977. .
Eoyang, et al. In: Proc in Nucleic Acids Research, vol. 2, pp.
829-839 (Cantoni & Davies, eds.) Harper & Row, N.Y., 1971.
.
L. Eoyang and J. T. August, In: Procedures In Nucleic Acids
Research, vol. 2, pp. 829-839, (Cantoni and Davies, eds.) Harper
and Row, New York (1971) ..
|
Primary Examiner: Sayala; Chhaya D.
Attorney, Agent or Firm: Galloway; Norval B.
Claims
I claim:
1. A method for purifying Q Beta replicase from cells in which it
is produced, comprising the steps of:
a) disrupting cells which produce Q Beta replicase to form a cell
lysate;
b) contacting the cell lysate with polyethyleneimine under
conditions appropriate for selective precipitation of nucleic acids
present in the lysate, resulting in formation of a supernatant
containing Q Beta replicase;
c) contacting the supernatant with a .[.anionic resin.].
.Iadd.cationic resin .Iaddend., under conditions appropriate for
binding of Q Beta replicase to the resin;
d) contacting the product of step (c) with a first elution buffer
under conditions appropriate for separation of Q Beta replicase
from the resin;
e) contacting the product of step (d) with an .[.cationic resin.].
.Iadd.anionic resin .Iaddend.under conditions appropriate for
binding of Q Beta replicase to the resin; and
f) contacting the resin of step (e) with a second elution buffer
under conditions appropriate for separation of Q Beta replicase
from the resin.
2. A method of claim 1, wherein the cells are E. coli cells which
produce Q Beta replicase.
3. A method of claim 1, wherein cells are disrupted in step (a) by
sonication or homogenization.
4. A method of claim 1, wherein the first elution buffer and the
second elution buffer comprise salt gradients having a
concentration of NaCl of from about 100 to about 400 mM.
5. A method of producing substantially pure Q Beta replicase,
comprising the steps of:
a) providing a lysate of Q Beta replicase-producing E. coli
cells;
b) contacting the cell lysate with a polyethyleneimine in the
presence of NaCl under conditions appropriate for selective
precipitation of nucleic acids present in the lysate, resulting in
formation of a supernatant containing Q Beta replicase;
c) contacting the supernatant with a .[.anionic resin.].
.Iadd.cationic resin .Iaddend.under conditions appropriate for
binding of Q Beta replicase to the resin;
d) contacting the product of step (c) with a first elution buffer
under conditions appropriate for separation of Q Beta replicase
from the resin;
e) contacting the product of step (d) with an .[.cationic resin.].
.Iadd.anionic resin .Iaddend.under conditions appropriate for
binding of Q Beta replicase to the resin; and
f) contacting the resin of step (e) with a second elution buffer
under conditions appropriate for separation of Q Beta replicase
from the resin.
6. A method of claim 5, wherein the polyethyleneimine is 10% (w/v)
polyethyleneimine.
Description
DESCRIPTION
Background
Q Beta replicase is an RNA dependent RNA polymerase that is
produced in E. coli as a result of infection by the bacteriophage Q
Beta. Q Beta replicase is composed of one phage-encoded polypeptide
of molecular weight 65,000, and three host-encoded polypeptides of
molecular weight 70 000, 45,000, and 35,000. These host-encoded
polypeptides are, respectively: 30 S ribsomal protein S1, and
protein synthesis elongation factors EF-Tu and EF-Ts. T. Blumenthal
and G. Carmichael, Ann. Rev. Biochem. 28:525-548 (1979).
Q Beta replicase autocatalytically replicates some specific types
of RNA known as variants in vitro. This RNA can be amplified
exponentially, to produce quantities of RNA which are then used in
bioassays. For example, midivariant (MDV) RNA produced by Q Beta
replicase in such an amplification system can be tagged with biotin
to form biotinylated RNA which combines with avidin to form an
adduct useful as a reporter for a variety of replication-assisted
bioassays for antibodies or nucleic acids. B. C. F. Chu et al.,
Nucleic Acids Res. 14:5591 (1986); E. A. Miele et al., J. Mol.
Biol., 171:281 (1983).
Q Beta replicase is generally obtained and isolated from Q Beta
phage-infected cells. Eoyang and August report a complex scheme for
isolating Q Beta replicase which consists of: 1) alumina extraction
of bacteriophage infected cells, 2) polyethylene glycol
(PEG)-dextran phase partitioning, 3) ammonium sulfate
precipitation, 4) phosphocellulose column chromatography, 5)
QAE-Sephadex column chromatography, 6) hydroxylapatite column
chromatography, and 7) Bio-Gel A.0.5 m column chromatography.
Eoyang, L. and August, J. L. Proc. Nucleic Acic Res. 829 (1972).
Kamen and others also describe schemes for obtaining Q Beta
replicase, which are variations of the above. Kamen, R. Biochem
Biophys. Acta. 262: 88-100 (1972); Sumper, M. and Luce, R. Proc.
Nat. Acad. Sci. USA, 162.166 (1975). Presently, available methods
of obtaining Q Beta replicase have several important limitations:
the resulting product is not sufficiently pure (i.e., contaminated
with MDV.sup.-1 RNA), the procedures are complex and labor
intensive, and they cannot be readily scaled up to a production
level to produce large quantities needed for use in commercial
assay systems. The use of alumina extraction and PEG-dextran phase
partitioning in the initial processing steps places severe
limitations on the scale of the process.
SUMMARY OF THE INVENTION
The present invention relates to a method or process for purifying
Q Beta replicase from cells which produce the enzyme and to highly
purified Q Beta replicase, obtained by the present method, which is
essentially free of midivariant or other template RNA contamination
(i.e., substantially pure Q B replicase). The method is a four-step
scheme which consists of 1) disrupting the cells to form a crude
cell lysate; 2) contacting the cell lysate with a polymeric agent
which selectively precipitates nucleic acids; 3) separating the
enzyme by chromatography; and 4) purifying the enzyme by
chromatography.
The subject method has several advantages that presently-available
methods do not offer. First, because the subject method includes a
step of disrupting the cells, kilogram amounts of cell paste can be
processed. In contrast, presently-available processes, such as
those including alumina extraction, are only useful for small
amounts, (i.e., about 100-200 grams) of cell paste. Second, because
it includes a precipitation step, the present method does not
include a PEG-dextran phase partitioning step, which is the major
rate limiting step in other processes. The use of precipitation in
the presence of sodium chloride in the present method not only
increases the amount of Q Beta replicase that is extracted from the
cells, but also eliminates several inhibitors of the enzyme that
are present in crude Q Beta replicase extracts made by other
processes. Third, through use of the present purification process,
high yields of the enzyme result. Fourth, the replicase produced
has a high level of specific activity, and nearly homogeneous
(nearly pure) enzyme is obtained. That is, the yield of Q Beta
replicase from the present process is nearly 10-fold higher than
yields from previously-available methods, the specific activity of
the product is nearly 8-fold higher than the specific activity of
the product of previously available methods; and Q Beta replicase
produced by the present method is nearly homogeneous, while enzyme
purified by prior art methods is only about 50% pure. Fifth, the
present process is simple and can ba readily scaled up. For
example, it can be used to produce highly purified Q Beta replicase
in sufficient quantities for use in a commercial Q Beta
amplification system.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows an SDS:PAGE gel comparing samples of purified Q Beta
replicase. Samples of reduced and denatured Q Beta replicase were
run on denaturing 10-15% polyacrylamide gradient gels, and stained
with Coomassie blue. Lane 1: Q Beta replicase purified by the
method of Eoyang and August, Proc. Nucleic Acid Res. 2:829 (1972).
Lane 2: Q Beta replicase purified by the present process. Lane 3:
Molecular weight markers.
FIG. 2 shows denaturing polyacrylamide gel analysis of RNA products
synthesized through the use of Q Beta replicase purified by the
present process. Lane 1:0 input molecules; Lane 2: 10.sup.2
molecules; Lane 3: 10.sup.4 molecules; Lane 4: 10.sup.8 molecules;
and Lane 5: MDV-FAL-St RNA marker.
FIG. 3 shows a denaturing polyacrylamide gel of RNA products
synthesized by Q Beta replicase purified by the method of Eoyang
and August, ibid. Lanes 1 and 2: 0 input molecules; Lane 3:
10.sup.2 molecules; Lane 4: 10.sup.4 molecules; Lane 5: 10.sup.6
molecules; Lane 6: 10.sup.8 molecules; and Lane 7: MDV-Fal-St.
FIG. 4 is a schematic representation of plasmid pTAC QB 6Bp
Kan.sup.r, which contains the Q Beta replicase gene under the
control of the tac promotor.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process or method for purifying
Q Beta replicase and to essentially pure or homogeneous Q Beta
replicase. The present method is useful for obtaining from cells
which produce Q Beta replicase (e.g., cells containing and
expressing a recombinant construct as well as phage-infected
cells), Q Beta replicase which is substantially free of
contaminants, such as midi-variant RNA, Q Beta RNA or other
template. As used herein, Q Beta replicase is said to be
"substantially free" of contaminants if, when it is incubated in
standard amplification assay buffer which does not contain any
additional RNA template, no detectable RNA systhesis occurs.
The present process has four general steps:
1) disruption of Q Beta replicase-producing cells, to produce a
(homogeneous) cell lysate;
2) separation of the enzyme from the cell lysate, which is carried
out, for example, by contacting the suspension with an amine, such
as polyethyleneimine resulting in precipitation of nucleic acids
and nucleic acid processing enzymes bound to them, and therefore,
initial separation of Q Beta replicase from unwanted cellular
materials;
3) contact of the supernatant produced in step (2) containing the
enzyme with a cationic resin, such as Q Sepharose; and
4) contact of the eluant produced in Step (3) with an anionic
resin, such as S Sepharose.
Through use of the present method, Q Beta replicase of greater
purity than can be obtained through previously known methods is
produced For example, Q Beta replicase shown to be more than 95%
pure, as judged by SDS polyacrylamide gel electrophoresis (PAGE),
has been obtained by the present method.
The present process is generally carried out at cold room
temperatures (about 4.degree. C.) to prevent the enzyme from
denaturing, although it is not necessary to do so. In the first
step of the present process, cells which produce Q Beta replicase
are disrupted. Any calls which produce Q Beta replicase can be used
as a source of Q Beta replicase. For example, the present method
can be used to obtain Q Beta replicase from bacterial cells which
have been infected with the Q Beta bacteriophage. Haruna and
Spiegelman, Proc. Nat'l Acad. Sci. U.S.A., 54:579 587 (1965). It
can also be used to obtain Q Beta replicase from cells which
contain a clone of the Q Beta replicase phage subunit, which
contains the Q Beta replicase gene under the control of an
appropriate promoter. The present method will now be described as
it has been used to purify Q Beta replicase from bacterial cells
(E. coli) expressing the cloned gene for the Q Beta replicase phage
subunit (constructed by D. Mills, Downstate Medical Center,
Brooklyn, NY) under the control of the promoter. It is to be
understood, however, that the present method can also be used to
purify Q Beta replicase from other cells in which it is produced.
Plasmid pTAC QB 6Bp Kan.sup.4, which contains the Q Beta replicase
gene under the control of the promoter is represented schematically
in FIG. 4. D. R. Mills, J. Mol. Biol., 200:489-500 (1988) U.S. Pat.
No. 4,786,600.
Q Beta replicase-producing cells are suspended in an aqueous medium
such as a buffer (e.g., Tris buffer), and treated to disrupt the
cells Cells can be disrupted by enzymatic methods (e.g., with
lysozyme) or by physical methods. Treatment which physically
disrupts the cells is generally by mechanical means, such as
sonication or homogenization. Such treatment causes the cell
membrane to rupture, resulting in release of the contents of the
cells into the suspending medium, forming a cell lysate.
In the second step of the present process, nucleic acids in the
cell lysate are selectively removed for example by contacting the
lysate with an agent which can selectively precipitate the nucleic
acids present in the lysate. In this step, the cell lysate is
contacted with the agent under conditions sufficient to cause
precipitation of nucleic acids and nucleic acid processing enzymes
while retaining Q Beta replicase in the supernatant. A particularly
useful agent for this purpose is polyethylenimine (PEI). However,
other agents which precipitate nucleic acids, such as streptomycin
sulfate, can be used. The precipitation step is necessary to remove
MDV--RNA, and other nucleic acids from the cell lysate. Once
precipitation is completed, the precipitate containing the PEI and
other cell debris is separated from the mixture, by centrifugation,
for example, and discarded. Q Beta replicase is present in the
resulting supernatant, which is subsequently treated in such a
manner, as described below, that substantially pure Q Beta
replicase is obtained.
In the third step of the present process, the supernatant is
diluted as needed (e.g., with a neutral buffer, such as Tris
buffer). The diluted material is then applied to a cationic
chromatography resin which binds Q Beta replicase enzyme The resin
is generally contained in a column. Q Sepharose is a particularly
useful resin for this purpose. However, other cationic resins, for
example, QAE Sephadex, can also be used. After the enzyme has been
loaded onto the resin, the resin is washed with several column
volumes of a neutral buffer, such as Tris, containing a low
concentration of a salt, such as NaCl. Once washing is complete,
the enzyme is eluted from the column using a salt gradient, which
ranges, for example, from about 100 to about 400 mM NaCl, in Tris
buffer. The fractions are collected, and tested for the presence of
the enzyme (e.g., by using an activity assay, such as the Poly C
assay described in the Exemplification). Fractions are selected in
order to minimize contaminants in the product. Preferably, the
fractions showing the highest incorporation rate (the "peak
activity" fractions) are pooled prior to the fourth step.
In the fourth step of the present process, the pooled fractions are
contacted with an anionic chromatography resin. S Sepharose is a
particularly useful resin for this purpose. The resin is preferably
contained in a column. The pooled fraction is diluted as needed to
lower the salt concentration (e.g., with the Tris buffer) and then
loaded onto the resin. Once all of the fraction has been loaded
onto the column, the column is washed, and then eluted with a salt
gradient, as in the third step. The eluant is assayed to determine
which fractions contain the peak enzyme activity.
Q Beta replicase obtained by this process is substantially
homogeneous, and has been shown to be free of MDV--.sup.1 RNA and
to be more than 95% pure, as measured by SDS PAGE. As described
below and in the Exemplification, the present method has been used
to obtain such substantially pure Q Beta replicase. The present
method can also be modified, however, to produce "less pure" enzyme
(i.e., Q Beta replicase in combination with a limited quantity of
other proteins), if desired. This can be effected by changing
(shortening) the length of the chromatography column used, by
increasing the quantity of materials loaded on a column, or by
using "step" gradients rather than linear gradients. The process is
conducive to scale-up to commercial production levels. The Q Beta
replicase recovered from the last step of the method is highly
active, and efficiently replicates midivariant (MDV) RNA templates
in vitro
In one embodiment of the present method, a culture of E. coli cells
infected with Q Beta replicase phage or cells containing a subunit
clone of the phage DNA encoding the 65,000 mw subunit of the enzyme
is used. The E. coli cells were transformed by a plasmid vector
which contains the Q Beta replicase gene under the control of the
tac promoter. The plasmid, designated pTAC QB 6Bp Kan, is shown
schematically in FIG. 4. E. coli cells transformed by the plasmid
produce Q Beta replicase. D. R. Mills, J. Mol. Biol. 200:489-500
(1988). The cells are suspended in a buffer or neutral salt
solution which contains 50 mM Tris.HCl (pH 7.8). 55 mM MgCl.sub.2,
5 mM 2.mercaptoethanol, 1 mM EDTA and 500 mM NaCl. This step, and
all of the subsequent steps in the process are carried out at
4.degree. C. The cells are disrupted by subjecting the cell
suspension to homogenization, for example, in a Matlin Gaulin
homogenizer, to form a crude cell lysate. The lysate is then
contacted with 10% (w/v) polyethyleneimine (PEI), causing
precipitation of the fraction containing MDV--.sup.1 RNA. The
precipitate and supernatant (which contains the Q Beta replicase)
are separated by centrifugation. The supernatant is diluted with a
buffer containing 50 mM Tris-HCl (pH 7.8), 5 mM MgCl.sub.2, 5 mM
2.mercaptoethanol and 1 mM EDTA. The diluted solution is then
contacted with Q Sepharose resin, which binds Q Beta replicase. The
column is eluted with a salt gradient containing from about 100 to
400 mM NaCl and the fractions collected and assayed for Q Beta
replicase activity. The fractions containing Q Beta replicase are
pooled, diluted with the same dilution buffer, and contacted with S
Sepharose resin. The Q Beta replicase selectively binds to the S
Sepharose resin, thereby making it possible to separate the enzyme
from other components. Q Beta replicase is eluted from the resin
using a salt gradient, as above. Enzyme is obtained in high purity
by this process.
The use of PEI precipitation followed by Q Sepharose and S
Sepharose column chromatography produces nearly homogeneous enzyme.
Other procedures require several additional steps to achieve the
same level of purity, with a lower overall yield. A comparison of Q
Beta replicase produced by the present process to enzyme produced
by the process of Eoyang, ibid, and August is shown in Table 1.
TABLE 1 ______________________________________ Comparison of Q Beta
Replicase Purified by the Method of Eoyang and August Versus
Present Method Process U/ml Total Units mg/ml Total mg U/mg
______________________________________ Eoyang and 173 1038 1.2 7.8
144 August Method* Present 670 20,100 0.59 17.7 1130 Method.sup.#
______________________________________ *Prepared from 200 g of
phageinfected cells .sup.# Prepared from 50 g of E. coli that
contain the Q Beta phage subuni gene under the control of the tac
promoter
A particularly valuable aspect of this process is that it yields
enzyme that is essentially free of MDV--.sup.1 RNA, whereas
replicase purified by the method of Eoyang and August is
contaminated with MDV--.sup.1 RNA.
The enzyme obtained through use of the present method efficiently
replicates Q Beta replicase templates, such as MDV RNA. Thus, the
enzyme is useful in bioassay systems (e.g., to produce MDV RNA for
use as a reporter group, as described by Chu et al. B. C. F. Chu et
al., Nucleic Acids Res., 14:5591 (1986)). Q Beta replicase produced
by the present process is also useful in systems for amplifying
RNA. The present enzyme, which is free of RNA contaminants and can
be more than 95% pure (as judged by SDS:PAGE), is particularly
useful in amplification and bioassy systems.
The invention is illustrated by the following exemplification,
which is not intended to be limiting in any way.
EXEMPLIFICATION
Purification of Q Beta Replicase from Phage-Infected E. Coli
Cells
One hundred (100 g) grams of recombinant E. coli cells containing
the cloned gene for the Q Beta replicase phage subunit under the
control of the tac promoter was used as the source of Q Beta
replicase.
The following procedure was carried out in a cold room (4.degree.
C.), and in an RNA-free environment, using sterile buffers to
minimize contamination with MDV.1 RNA. The E. coli cells were
suspended in a one liter beaker in 2.5 volumes of a solution
containing 50 mM Tris-HCl buffer, (pH 7.8), 55 mM MgCl.sub.2, 5 mM
2. mercaptoethanol, 1 mM (ethylenediaminetetraacetic acid) EDTA and
500 mM NaCl. A clean stir bar was added and the contents of the
beaker were stirred at 4.degree. C. until the suspension was
homogeneous
One half of the cell suspension was transferred to a Rosett
sonicating cell (250 ml Heat Systems-Ultrasonics) and the cell was
placed in an ice water bath. The remainder of the cell suspension
was kept on ice.
The cell suspension was sonicated 4 times, 2 minutes each time, at
an output control setting of 7. After each 2 minute interval, the
temperature of the suspension increased from 4.degree. C. to about
10.degree.-12.degree. C. The cell suspension was allowed to cool
down to 6.degree. C. or less before resuming sonication. After
sonication was complete, the volume of the sonicated cell
suspension was measured. The suspension was transferred to a clean
1 L beaker and placed on ice. The procedure was repeated with the
remainder of cell suspension.
A clean stir bar was added to the beaker containing sonicated cell
suspension and the contents stirred on a stir plate at 4.degree. C.
0.03 volumes of 10% polyethyleneimine (w/v) (Aldrich) was gradually
added to the cell homogenate.
After all the polyethyleneimine was added, the suspension was
stirred for an additional 15 minutes.
The contents of the beaker were transferred to two 250 ml
polypropylene centrifuge bottles and centrifuged in a sorvall GSA
rotor for 30 minutes at 10,000 rpm at 4.degree. C. The clear
supernatant was removed from the pellet material. The volume of the
supernatant was measured and transferred to a clean 2 L beaker.
The supernatant was diluted with 4 volumes of the solution of 50 mM
Tris-HCl (pH 7.8) 5 mM MgCl.sub.2, 5 mM 2.mercaptoethanol, 1 mM
EDTA, and applied in a concentration of 30 mg of protein per ml of
resin to an equilibrated 220 ml Q Sepharose column (Q Sepharose
Fast Flow resin, Pharmacia) at a flow rate of 400-450 ml/hr. A UV-1
monitor (Pharmacia, 280 nm filter) was set at a AUFS of 2.0 and the
chart speed of 0.2 mm/min.
While the enzyme was being loaded onto the column, the supernatant
fraction was assayed for Q Beta replicase activity using the Poly C
assay (described below).
After all of the enzyme had been loaded onto the the column was
washed with a solution of 50 mM Tris-HCl (pH 7.8), 5 mM MgCl.sub.2,
5 mM 2. mercaptoethanol, 1 mM EDTA, and 100 mM NaCl, at a flow rate
of 400-450 ml/hr. The column was washed until the UV absorbance
decreased to less than 0.4 (4.6 column volumes).
A 10 column-volume (2.times.1100 ml) gradient was prepared ranging
from 100.400 mM NaCl in 50 mM Tris.HCl (pH 7.8), 5 mM MgCl.sub.2, 5
mM 2.mercaptoethanol, 1 mM EDTA, and the gradient was run at a flow
rate of 200-225 ml/hr. Fractions of 22 ml were collected. The chart
speed of the recorder was increased to 0.5 mm/min.
To minimize the risk of contaminating the fractions with MDV RNA,
0.1-0.2 ml aliquots of the fractions to be assayed were removed
using sterile 1 ml pipettes and transferred to serile 1.5 ml
microcentrifuge tubes.
The Q Sepharose fractions were assayed for Q Beta replicase
activity using the Poly C assay.
The peak replicase fractions were assayed for the presence of
contaminating RNase, and for activity in the absence of any
template to determine whether any of the peak fractions contained
MDV.1 RNA.
The peak Q Beta replicase fractions were determined based on the
following criteria:
1) fractions that contain 50% +/5% of the activity of the maximum
fraction;
2) fractions that are free of major RNase contamination; and
3) fractions that are free of MDV-1 RNA contamination.
Fractions meeting these criteria were pooled.
The protein concentration of the fractions were determined using
the Bradford Assay. M. Bradford, Anal. Biochem., 72:248 (1976).
Replicase activity was determined using the Poly C assay.
An S Sepharose (Pharmacia) column was poured and equilibrated with
10 column volumes of 50 mM Tris-HCl (pH 7.8) 5 mM MgCl2. 5 mM
2.mercaptoethanol, 1 mM EDTA, and 100 mM NaCl.
Once the column was equilibrated, the pooled fraction was diluted
with 2.5 volumes of 50 mM Tris-HCl (pH 7.8) 5 mM MgCl.sub.2, 5 mM
2.mercaptoethanol, 1 mM EDTA. The diluted enzyme was applied to the
column in a concentration of 10 mg of protein per ml of resin at a
flow rate of two column volumes per hour. The UV-1 monitor was set
at an AUFS setting of 0.5 and the chart speed at 0.2 mm/min.
Once all of the enzyme was applied to the column, the column was
washed with 50 mM Tris-HCl (pH 7.8), 5 mM MgCl.sub.2, 5 mM
2.mercaptoethanol, 1 mM EDTA, and 100 mM NaCl and a 10 column
volume gradient of 100 mM to 400 mM NaCl was run at a flow rate of
1 column volume per hour. Fractions of 0.01 volume of the total
gradient were collected. The chart speed of recorder was increased
to 0.5 mm/min.
To minimize the risk of contaminating the column fractions with MDV
RNA, 0.1 ml aliquots were removed using sterile 1 ml pipettes and
transferred to sterile 1.5 ml microcentrifuge tubes.
The S Sepharose fractions were assayed for replicase activity using
the Poly C assay, to define the location of the replicase peak.
To discriminate between the enzyme fractions which contain Q Beta
"Holoenzyme" versus those that contain Q Beta "alpha-less" enzyme
which does not have the S1 subunit and cannot replicate MDV RNA
templates, aliquots of the peak fraction were run on denaturing
10-15% poly.
Fractions containing the highest replicase activity were assayed
for the presence of contaminating RNases, and were assayed in the
absence of any added template to determine whether any of the peak
fraction contained MDV.1 RNA.
The "peak" fractions were determined based on the following
criteria:
1) fractions that contain at least 33% +/5% of the activity of the
maximum fraction;
2) fractions that contain only Q Beta "Holoenzyme";
3) fractions that are free of RNase contamination; and
4) fractions that are free of MDV.1 RNA.
Fractions meeting all four of these criteria were pooled. The
protein concentration of this fraction was determined using the
Bradford Assay. Replicase activity was determined using the Poly C
assay.
The fraction was also assayed for replicase activity using MDV RNA
as a template.
An equal volume of chilled ultra pure glycerol was added to the
remainder of the fraction, and mixed gently until no Schleiren
lines were observed.
Q Beta Replicase Assay-Poly C Template
A reaction mixture was prepared by combining the following reagents
in a 1.5 ml micro-centrifuge tube at room temperature:
______________________________________ 5 .mu.l of 5 .times. Q beta
replicase buffer 5 .mu.l of 1.0 mg/ml Poly C (Sigma) 1 .mu.l of 10
mM GTP (Pharmacia) 1 .mu.l of 50 .mu.g/ml Rifampicin (Sigma) 0.5
.mu.l of alpha-.sup.32 P GTP, 10.mu. Ci/.mu.l (New England Nuclear)
11.5 .mu.l DEPC treated water 1 .mu.l of enzyme 25 .mu.l total
volume ______________________________________
The 5.times.Q Beta replicase buffer consisted of 450 mM Tris-HCl,
(pH 7.8), and 70 mM MgCl.sub.2.
After all reagents, except the enzyme, had been combined, the tube
was vortexed for 10 seconds and then centrifuged at 12,000.times.g
for 2 seconds to get all of the reagents to the bottom of the
tube.
The enzyme was added; the tube was mixed gently and incubated for
10 minutes at 37.degree. C.
10 .mu.l aliquots were removed from each reaction tube and pipetted
onto DE.81 filters (Whatman). The filters were air dried for 1.2
minutes.
From one of the reaction tubes, 10 .mu.l was removed and pipetted
onto a DE.81 filter, allowed to air dry, placed in a scintillation
vial containing 3 ml of water and counted. From this data the total
number of counts that were added to each tube could be
determined.
The filters were transferred to a 1 L beaker containing 200 ml of
sodium phosphate (500 mM, pH 7.4) wash buffer. The filters were
washed for 20 minutes at room temperature with occasional shaking.
The wash solution was decanted and an additional 200 ml of fresh
wash buffer solution was added to the beaker and the filters were
washed again. The wash step was for 20 minutes. The buffer was
removed, and repeated one more time. After the third wash, 200 ml
of distilled, deionized water was added to the beaker and the
filters were washed for 10 minutes with mixing.
The filters were removed from the water and blotted dry on a piece
of Whatman 3 mm paper.
The filters were transferred to scintillation vials (Wheaton)
containing 3 ml of water.
The vials were capped and the radioactivity in each vial was
determined in a scintillation counter at settings for detection of
.sup.32 P, to determine the level of incorporation by each
fraction.
Q Beta Replicase Assay-MDV RNA Template
A reaction mixture was prepared by combining the following reagents
in a 1.5 ml microcentrifuge tube at room temperature:
______________________________________ 5 .mu.l of Q Beta replicase
buffer 4 .mu.l of a mixture containing 2.5 mM each of ATP, CTP, UTP
and GTP (Pharmacia) 1 .mu.l of alpha-.sup.32 P GTP, 10.mu. Ci/.mu.l
(New England Nuclear) 9 .mu.l of DEPC treated water 19 .mu.l per
assay tube ______________________________________
After the reagents had been combined, the tube was vortexed for 10
seconds and centrifuged at 12,000.times.g for 2 seconds to get all
of the contents to the bottom of the tube.
5 .mu.l of an appropriate MDV RNA dilution was added to the above
reaction mix. To minimize the risk of contamination, a separate
micropipettor was used to dispense the MDV RNA solution. To those
reactions where no RNA is added, 5 .mu.l of DEPC-treated water was
added.
1 .mu.l of Q Beta replicase was added to above reaction mix, which
was mixed gently and incubated 20 minutes at 37.degree. C.
After 20 minutes, 2 .mu.l of reaction was removed and added to 18
.mu.l of 120 mM NaCl --20 mM EDTA and placed on ice.
From one of the reaction tubes, 10 .mu.l was removed and pipetted
onto DE-81 filter, allowed to air dry, placed in a scintillation
vial contain 3 ml of water, and counted. From this data the total
number of counts added to each reaction could be determined.
10 .mu.l of the 20 .mu.l sample from the previous step was pipetted
onto a DE-81 filter. The remainder was saved for gel analysis.
The filters were dried for 1-2 minutes, washed and counted as
described above for the Poly C assay.
To analyze amplification reaction products on a denaturing
polyacrylamide gel, a 6% poly-acrylamide 7 M urea gel was poured
and allowed to polymerize The gel was pre-run at 50 W (constant
power) for 30 minutes.
1 .mu.l of samples were added to 3 .mu.l of Formamide-Dye loading
buffer. The samples were placed in a boiling water bath for 3
minutes, and transferred to ice until loading onto gel.
The samples were run at 50 W (constant power) until the Bromphenol
Blue marker had run off the gel.
The gel was dismantled and placed in a cassette on film (Kodak
XAR5) overnight at -70.degree. C. with an intensifying screen. The
film was developed in an automatic film processor.
The activity of Q Beta replicase produced by the present method is
summarized in Table 2.
TABLE 2 ______________________________________ Summary of Q Beta
Replicase Purification Fraction Units/ml Total Units mg/ml Total mg
U/mg ______________________________________ Crude Lysate 190 24,700
23.5 3035 8 Q Sepharose 1013 69,900 4.25 293 238 S Sepharose 670
20,100 0.59 17.7 1130 ______________________________________ 1 U =
1 nmol of GMP incorporated in 10 min at 37
Table 2 shows the recovery of enzyme at each step in the
purification process (from 50 g of cells).
The enzyme produced by the present method was incubated in standard
amplification assay buffer (90 mM Tris-HCl (pH 7.8) and 14 mM
MgCl.sub.2) which did not contain any added RNA template and no
detectable RNA synthesis was observed after 20 minutes of
incubation at 37.degree. C. (FIG. 2, lane 1). MDV.1 RNA was not
observed even after 60 minutes of incubation.
Amplification reactions containing the present enzyme were primed
with 10.sup.2 -10.sup.8 MDV-Fal-St molecules, (P. M. Lizardi et
al., Biotechnology, 6:1197-1202 (1988(, and the only product
observed was full length MDV-Fal-St (FIG. 2, lanes 2-4). However,
when enzyme prepared by the method of Eoyang and August was
incubated under the same conditions as above, RNA synthesis was
observed in the absence of added RNA template, indicating
contamination of the enzyme preparation with MDV.1 RNA.
The resulting material was analyzed on a denaturing polyacrylamide
gel, and as shown in FIG. 3, MDV.1 RNA (FIG. 3, lanes 1 and 2) was
produced. Furthermore, when amplification reactions containing this
enzyme were primed with 10.sup.2 -10.sup.8 MDV-Fal-St molecules,
both and Fal-St and MDV-1 RNA were synthesized (FIG. 3, lanes
3-6).
Equivalents
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described specifically
herein. Such equivalents are intended to be encompassed in the
scope of the following claims.
* * * * *