U.S. patent application number 14/567368 was filed with the patent office on 2015-06-04 for mutant proteinase with reduced self-cleavage activity and method of purification.
The applicant listed for this patent is YALE UNIVERSITY. Invention is credited to Robert T. Batey, Jennifer A. Doudna, Louise J. Lucast.
Application Number | 20150152403 14/567368 |
Document ID | / |
Family ID | 22787327 |
Filed Date | 2015-06-04 |
United States Patent
Application |
20150152403 |
Kind Code |
A1 |
Doudna; Jennifer A. ; et
al. |
June 4, 2015 |
Mutant Proteinase with Reduced Self-Cleavage Activity and Method of
Purification
Abstract
The present invention provides a mutant 27 kDa NIa proteinase
having reduced self-cleavage activity relative to the self-cleavage
activity of its wild-type proteinase. The mutant has the same
substrate cleavage activity as the wild-type proteinase but is more
stable than the wild-type proteinase. The present invention also
provides a method of obtaining large quantities of active 27 kDa
NIa proteinase for use as a tool for purification of other
proteins.
Inventors: |
Doudna; Jennifer A.;
(Berkeley, CA) ; Lucast; Louise J.; (Guilford,
CT) ; Batey; Robert T.; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YALE UNIVERSITY |
NEW HAVEN |
CT |
US |
|
|
Family ID: |
22787327 |
Appl. No.: |
14/567368 |
Filed: |
December 11, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13898650 |
May 21, 2013 |
8969062 |
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14567368 |
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13482535 |
May 29, 2012 |
8470575 |
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13898650 |
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12184315 |
Aug 1, 2008 |
8206968 |
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13482535 |
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10343766 |
Oct 9, 2003 |
7494786 |
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PCT/US01/18620 |
Jun 11, 2001 |
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12184315 |
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60211535 |
Jun 15, 2000 |
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Current U.S.
Class: |
435/68.1 ;
435/219; 435/252.31; 435/252.33; 435/252.35; 435/254.11; 435/254.2;
435/254.21; 435/320.1; 435/325; 435/348; 435/352; 435/356; 435/357;
435/358; 435/364; 435/365; 435/367; 435/419; 536/23.2 |
Current CPC
Class: |
C07K 2319/00 20130101;
C12P 21/06 20130101; C07H 21/04 20130101; C12Y 304/22044 20130101;
C07K 2319/21 20130101; C12N 9/506 20130101 |
International
Class: |
C12N 9/50 20060101
C12N009/50; C12P 21/06 20060101 C12P021/06 |
Claims
1. An isolated mutant proteinase having a molecular weight of about
27 kDa and reduced self-cleavage activity relative to the
self-cleavage activity of its wild-type proteinase.
2. The mutant proteinase of claim 1, wherein the mutant proteinase
comprises an amino acid sequence in which the residue corresponding
to Ser 219 of the 27 kDa NIa proteinase is replaced with another
residue.
3. The mutant proteinase of claim 2, wherein the residue replacing
Ser 219 of 27 kDa NIa proteinase is Asn.
4. A composition comprising the mutant proteinase of claim 1 and a
carrier.
5. A fusion protein comprising the mutant proteinase of claim 1
fused to a heterologous polypeptide.
6. The fusion protein of claim 5, wherein the heterologous
polypeptide consists of six histidines.
7. An isolated nucleic acid molecule comprising a sequence encoding
a mutant proteinase having a molecular weight of about 27 kDa and
reduced self-cleavage activity relative to the self-cleavage
activity of its wild-type proteinase.
8. An isolated nucleic acid molecule of claim 7, wherein the
nucleic acid molecule encodes an amino acid sequence in which the
residue corresponding to Ser 219 of the 27 kDa NIa proteinase is
replaced with another amino acid.
9. An isolated nucleic acid of claim 8, wherein the nucleic acid
comprises a sequence that encodes an amino acid sequence in which
the residue corresponding to Ser 219 of the 27 kDa NIa proteinase
in Asn.
10. A vector comprising the nucleic acid of claim 7.
11. An expression vector comprising the nucleic acid of claim
7.
12. A host cell comprising the expression vector of claim 11.
13. A method of producing a proteinase having a molecular weight of
about 27 kDa and reduced self-cleavage activity relative to the
self-cleavage activity of its wild-type proteinase activity
comprising cultivating the host cell of claim 12 under conditions
that allow expression of the proteinase.
14. a nucleic acid encoding a fusion protein comprising a
proteinase, having a molecular weight of about 27 kDA and reduced
self-cleavage activity relative to the self-cleavage activity of
its wild-type proteinase, fused to a heterologous protein.
15. The nucleic acid of claim 14, wherein the heterologous protein
consists of six histidines.
16. A method of purifying a polypeptide that forms inclusion bodies
in a cell comprising the following steps: a) obtaining cells
expressing the polypeptide; b) lysing the cells; c) pelleting
inclusion bodies by centrifugation of the lysed cells; d)
centrifuging the inclusion bodies; e) solubilizing the inclusion
bodies in buffer containing solubilization f) centrifuging the
solubilized inclusion bodies to obtain a supernatant containing the
polypeptide; g) loading the supernatant over a single denaturing
column; h) collecting the proteinase; and i) renaturing the
collected polypeptide.
17. The method of claim 16, wherein the polypeptide has a molecular
weight of about 27 kDa and reduced self-cleavage activity relative
to its wild-type polypeptide.
18. The method of claim 16, wherein the polypeptide is selected
from the group consisting of tobacco etch virus (TEV) 27 kDa NIa
proteinase and mutant TPSN 27 kDa NIa proteinase.
19. The method of claim 16, wherein in step (g) the denaturing
column contains Ni-NTA.
20. A method of using the proteinase of claim 1, comprising
incubating the proteinase with a protein for a sufficient amount of
time to allow cleavage of the protein.
Description
FIELD OF THE INVENTION
[0001] The present invention is related to compositions comprising
mutant proteinase having reduced self-cleavage activity relative to
the self-cleavage activity of its wild-type proteinase. The mutant
proteinase has the same substrate cleavage-activity as its
wild-type proteinase and it is more stable than its wild-type
proteinase. The present invention is also related to methods of
obtaining large quantities of purified active proteins that form
inclusion bodies in cells.
BACKGROUND
[0002] Proteinases are present in both prokaryotic and eukaryotic
systems and have been shown to play an important role in the
processing of large precursor polyproteins during viral
replication. The reliance of viruses on proteolytic processing by
virally encoded proteases has been suggested to have several
evolutionary advantages, such as a need for reduced genomic content
and partial release from the constraints of the mechanisms of
transcriptional and translational regulation of the host cell
(Lawson et al., 1990). Viruses have evolved methods for regulating
the proteolytic cascade that produces viral strucural and
replication proteins to replace the host mechanisms. Lawson et al.
(1991) report that in picornaviruses, the temporal and spatial
distribution of expressed protease activity affects the appearance
and location of the final proteolytic product.
[0003] Polyviruses are members of the picornaviral family,
polyviruses make extensive use of proteinases during replication.
An example of a potyvirus that possesses a genome encoding a single
large polyprotein proteolytically processed by virally encoded
protease is the tobacco etch virus (TEV). TEV has a single-strand,
plus-sense RNA genome of about 9,500 nucleotides. The RNA is
organized as a single open reading frame and encodes a 346 kDa
polyprotein (Allison et al., 1986). The polyprotein is co- and
post-translationally processed by viral encoded proteinases. Two
proteinases, P1 and helper component proteinase (HC-Pro), are
responsible for their autocatalytic release from the amino-terminus
of the polyprotein (Verchot et al. 1991; Carrington et al., 1989).
The third proteinase, nuclear inclusion proteinase (NIa), mediates
all other cleavage events.
Characterization of the 27 kDa NIa Proteinase of Polyvirus
[0004] The NIa proteinase is 49 kDa and is found as an aggregate
with the 54 kDa NIb polypeptide in nuclear inclusion bodies in
infected plant cells (Carrington et al., 1988; Parks et al., 1995).
The 49 kDa NIa proteinase is a picornavirus 3C-like proteinase that
recognizes cleavage sites within the C-terminal two-thirds of the
polyprotein. The proteolytic domain of NIa lies within the
C-terminal half of the protein and has a molecular weight of about
27 kDa, while the N-terminal region of NIa comtains the Vpg (viral
protein, genome-linked) activity and has a molecular weight of
about 21 kDa.
[0005] Structurally, the 27 kDa NIa proteinase has been reported to
be similar to the trypsin-like family of cellular serine
proteinases, such as chymotrypsin or trypsin, with the substitution
of Cys for aerine as the active site nucleophile (Blazan et al.,
1990; Dougherty et al., 1989). Dougherty et al. (1989) disclose
that the catalytic triad of 27 kDa proteinase is composed of His,
Asp, and Cys, being similar to the catalytic triad found in other
viral proteinases (Dougherty et al., 1989). However, unlike the
other proteinases, the 27 kDa proteinase recognizes an extended
heptapeptide sequence, E-X-X-Y-X-Q.dwnarw.S/G (positions
P6-P1.dwnarw.P'1; X is any amino acid) (SEQ ID NO:1), and cleaves
within the heptapeptide sequence (Dougherty et al., 1989a;
Dougherty et al., 1988; Dougherty et al., 1989b). Residues at
positions P6, P3, P1, and P'1 are conserved aend essential for
optimal cleavage. Amino acids at the other positions appear to
modulate the rate at which cleavage occurs (Dougherty et al., 1989;
Dougherty and Parks, 1989).
[0006] Moreover, the 27 kDa NIa proteinase appears to be
structurally and functionally similar to other plus-stranded RNA
viral-encoded proteinases (Kransslich and Wimmer, 1998). First of
all, it cleaves the polyprotein between particular Gln-Gly or
Glyn-Ser dipeptides. Secondly, proteolytic activity is enhanced by
dithiothreitol. Thirdly, the gene encoding this proteinase is
adjacent to the putative RNA-dependent, RNA-polymerase gene.
Lastly, the proteinase contains a conserved C-terminal amino acid
motif (Cys--15 amino acid--His) (Argos et al., 1984). This last
characteristic is shared by proteinases encoded by many RNA viruses
that translationally express their genetic information as a single
polyprotein from genome length RNA (Dougherty et al., 1989).
[0007] Addditionally, Parks et al. (1995) report that the 27 kDa
NIa proteinase contains an internal self-cleavage site positioned
at 24 amino acids from the carboxyl terminus of the proteinase and
that the active 27 kDa proteinase converts to a lower molecular
weight form with time. The 27 kDa NIa proteinase lacking the
C-terminal 24 amino acids exhibits limited activity. The truncated
proteinase is about one-twentieth as efficient in proteolysis of a
test peptide substrate as the full length form, and Parks et al.
(1995) indicate that the 27 kDa NIa proteinase appears to lose its
activity with time.
[0008] Further, Polayes et al. (1994) disclose that the 27 kDa NIa
proteinase is a highly specific protease that is active under a
broad temperature range and on a variety of substrates. Polayes et
al. report rapid cleavage at 30.degree. C. and 37.degree. C., about
80% cleavage at both 21.degree. C. and 16.degree. C. in one hour,
and 50% cleavage at 4.degree. C. Accordingly, Polayes et al.
recommend the use of this proteinase as a tool for removing
affinity tags from fusion proteins.
Use of 27 kDa NIa Proteinase Cleavage System for Purification of
Proteins
[0009] Parks et al. (1994) disclose an improved method for the
production, cleavage, and purification of fusion proteins and
peptids using the 27 kDa NIa proteinase. The method comprises
producing a fusion protein comprising the protein of interest, a
carrier peptide (such as an affinity carrier) and a 27 kDa NIa
proteinase cleavage site inserted between the two, purifying the
fusion protein, and incubating the fusion protein with the 27 kDa
NIa proteinase to remove the carrier peptide from the protein of
interest.
[0010] Johnston et al., U.S. Pat. No. 5,532,142, disclose a similar
method of isolation and purification of recombinant proteins using
the 27 kDa NIa proteinase. Like the purification method of Parks et
al. (1994), the method of Johnston et al. involves producing large
quantities of the fusion protein containing a desired protein fused
to the 27 kDa NIa proteinase cleavage site which is the carrier
peptide, purifying the fusion protein, and incubating the purified
fusion protein with the 27 kDa NIa proteinase to remove the carrier
peptide from the desired protein.
[0011] Unlike other proteinases, the 27 kDa proteinase exhibits
high specificity, insensitivity to many proteinase inhibitors used
in protein purification, and efficient cleavage under a broad range
of temperatures (Polayes et al., 1994). Moreover, the protein of
interest ie easily separated from the carrier peptide and the 27
kDa proteinase. For these reasons, there is an on-going interest in
obtaining large quantities of the active protein for use as a tool
in protein purification.
Purification of Proteins Including the 27 kDa NIa Proteinase from
Inclusion Bodies
[0012] Purification of Proteins that Form Inclusion Bodies. The
development of recombinant DNA technology has enabled the cloning
and expression of proteins in bacteria, yeast and mammalian cells
and has made it possible to produce therapeutics and industrially
important proteins at economically feasible levels. However, the
expression of high levels of recombinant proteins in Escherichia
coli ofen results in the formation of inactive, denatured protein
that accumulates in intracellular aggregates known as insoluble
inclusion bodies (Krueger et al., "Inclusion bodies from proteins
produced at high levels in Escherichia coli," in Protein Folding,
L. M. Gierasch and P. King (Eds), Am. Ass. Adv. Sci., 136-142
(1990); Marston, Biochem, J. 240:1-12 (1986); Mitraki et al.,
Bio/Technol. 7: 800-807 (1989); Schein, Bio/Technol. 7:1141-1147
(1989); Taylor et al., Bio/Technol, 4: 553-557 (1986)). Inclusion
bodies are dense aggregates, which are 2-3 m in diameter and
largely composed of recombinant protein, that can be separated from
soluble bacterial proteins by low-speed centrifugation after cell
lysis (Schoner et al., Biotechnology 3:151-154 (1985)).
[0013] The recovery of recombinantly expressed protein in the form
of inclusion bodies has presented a number of problems. First,
although the inclusion bodies contain a large percentage of the
recombinantly produced protein, additional contaminating proteins
must be removed in order to isolate the protein of interest.
Second, the proteins localized in inclusion bodies are in a form
that is not biologically active, presumably due to incorrect
folding.
[0014] Several methods have been developed to obtain active
proteins from inclusion bodies. These strategies include the
separation and purification of inclusion bodies from other cellular
components, solubilization and reduction of the insoluble material,
purification of solubilized proteins and ultimately renaturation of
the proteins and generation of native disulfide bonds. The art
teaches that concentrations of 6 M or greater of chaotropic agents,
such as guanidine hydrochloride, guanidine isothiocyanate or
urea--are necessary for solubilization of the insoluble recombinant
polypeptides from the inclusion bodies. See, for example,
Vandenbroeck et al. Eur. J. Biochem. 215;481-486 (1993); Meagher et
al., Biotech. Bioeng. 43:969-977 (1994); Yang et al., U.S. Pat. No.
4,705,848, issued Nov. 10, 1987; Weir et al., Biochem. J. 245:85-91
(1987); and Fischer Biotech. Adv. 12:89-101 (1994). However, the
use of high concentration of chaotropic agents, such as guanidine
hydrochloride, to solubilize proteins denatures the proteins.
[0015] U.S. Pat. No. 5,912,327 discloses the use of low
concentrations of guanidine salts, about 0.7 to about 3.5 M, to
solubilize biologically active (i.e., correctly folded) proteins
and extract this population of the protein from a heterogenous
protein mixture localized in inclusion bodies. The method described
in the patent comprises releasing the inclusion bodies containing
the target protein from the cells by lysis, optionally washing the
cells to remove cellular components, extracting with solutions
containing low concentration of guanidine salts, refolding target
proteins which have been solubilized using guanidine salts by rapid
dilution of guanidine salt extracts and optionally employing agents
which facilitate target protein refolding. The protein can then be
recovered and purified by methods well known to the skilled
artisan. However, this method is labor intensive.
[0016] Tissue plasminogen activator (iPA or TPA) is one example of
a pharmaceutically important drug produced by recombinant methods.
Unfortunately the current methods for producing tPA from bacterial
cell culture are both costly and laborious. The product of tPA in
heterologous host organisms relies on the production of inactive
tPA intracellularly in inclusion bodies, and the subsequent
isolation and purification of such inclusion bodies, followed by
activation of the tPA once freed from the inclusion bodies. U.S.
Pat. No. 5,077,392 discloses a renaturation method for refolding
denatured proteins obtained after expression in inclusion bodies.
tPA is isolated as a denatured reduced protein and on subsequent
oxidation refolded under oxidizing conditions to obtain what was
reported as up to a 26% yield of "reactivated" protein. While the
method appeared to improve polypeptide yield, the process involves
multiple, time-consuming steps, due to the initial recovery of the
insoluble, inactive protein.
[0017] Purification of 27 kDa NIa Proteinase. The 27 kDaNIA
proteinase has been especially difficult to isolate and purify in
large quantities and in active form because of its proclivity to
form inclusion bodies in nature. Previously published purification
protocols of TEV nuclear inclusion bodies from infected plant
tissue have demonstrated considerable proteolytic activity
(Dougherty et al., 1980). However, attempts to separate the 49 kDa
NIa proteinase from the NIb protein and other components and to
purify the NIa proteinase have resulted in loss of protein activity
(Parks et al., 1995).
[0018] Parks et al. (1995) describe purification of the soluble
fraction of recombinantly produced 27 kDa NIa proteinase. The
purification method of parks et al. involves overexpressing the
recombinant form of the proteinase as a fusion protein comprising a
seven-His tag at the N-terminus and purifying the fusion protein
using two separate columns, a nickel-nitrilotriacetic acid-agarose
(Ni-agarose) column and a cation-exchange column. This method is
labor-intensive and produces insufficient quantities of proteinase
for use as a general tool in protein purification.
[0019] Johnston et al., U.S. Pat. No. 5,532,142, discloses
recombinant vectors for overproducing plant virus proteinases in
suitable hosts. Johnston et al. use the same purfication protocol
as that of Parks et al. (1995) to purify the 27 kDa NIa proteinase.
The yield of purified proteinase is typically in the range of 5
mg/liter of cell culture and not all of it is active. Thus, the
yield of active protein is very low.
[0020] As discussed above, the 27 kDa NIA proteinase contains an
internal self-cleavage site that when cleaved, produces a
proteinase with reduced substrate cleavage activity. At the
present, there is no known method of stabilizing the proteinase,
and there is no known method of obtaining large quantities of
purified active 27 kDa NIa proteinase in large quantities.
Accordingly, there is a need to develop a method of obtaining large
quantities of purified active 27 kDa proteinase that will not
cleave itself.
SUMMARY OF THE INVENTION
[0021] The present invention provides mutant proteinases having a
molecular weight of about 27 kDa and reduced self-cleavage activity
relative to the self-cleavage activity of its wild-type proteinase.
In one embodiment, the mutant proteinases of the present invention
comprise an amino acid sequence in which the residue corresponding
to Ser 219 of the wild-type 27 kDa NIa proteinase is replaced with
another residue. In another embodiment, the mutant proteinases of
the present invention comprises an amino acid sequence in which the
residue corresponding to Ser 219 is replaced with Asn.
[0022] The present invention also provides composition comprising
the mutant proteinase. Preferably, the composition comprises a
carrier in addition to the mutant.
[0023] Moreover, the present invention provides fusion proteins
comprising mutant proteinase having a molecular wiehgt of about 27
kDa and reduced self-cleavage activity, fused to a heterologous
polypeptide, fusion partner, or carrier protein. In one embodiment,
the heterologous polypeptide, fusion partner, or carrier protein
comprises a protein that facilitates its isolation. In a preferred
embodiment, the heterologous polypeptide consists of six
histidines.
[0024] The present invention includes nucleic acid molecules
comprising a sequence encoding a mutant proteinase having a
molecular weight of about 27 kDa and reduced self-cleavage activity
relative to the self cleeavage activity of to its wild-type
proteinase. In one embodiment, the nucleic acid molecules encode
mutant proteinases comprising an amino acid sequence in which the
residue corresponding to Ser 219 of the 27 kDa NIa proteinase is
replaced with another amino acid. In a preferred embodiment, the
nucleic acid molecules encodes mutant proteinases comprising an
amino acid sequence in which the residue corresponding to Ser 219
is replaced with Asn.
[0025] The present invention also includes vectors, expression
vectors, and host cells comprising a nucleic acid molecule encoding
a mutant proteinase having a molecular weight of about 27 kDa and
reduced self-cleavage activity relative to the self-cleavage
activity of its wild-type proteinase.
[0026] Further, the present invention includes nucleic acid
molecules encoding fusion proteins comprising a proteinase, having
a molecular weight of about 27 kDA and reduced self-cleavage
activity relative to the self cleavage activity of its wild-type
proteinase, fused to a heterologous protein.
[0027] The present invention provides methods of producing a
proteinase having a molecular weight of about 27 kDa and reduced
self-cleavage relative to the self-cleavage activity of its
wild-type proteinase comprising cultivating a host cell comprising
a nucleic acid encoding the proteinase under conditions that allow
expression of the proteinase.
[0028] The present invention also provides a method of purifying a
polypeptide that forms inclusion bodies in a cell comprising:
[0029] a) obtaining cells expressing the polypeptide; [0030] b)
lysing the cells; [0031] c) pelleting inclusion bodies by
centrifugation of the lysed cells; [0032] d) centrifuging the
inclusion bodies; [0033] e) solubilizing the inclusion bodies in
buffer containing solubilization agent; [0034] f) centrifuging the
solubilized inclusion bodies to obtain a supernatant containing the
polypeptide; [0035] g) loading the supernatant over a single
denaturing column; [0036] h) collecting the proteinase; and [0037]
i) renaturing the collected polypeptide.
[0038] In one embodiment, the polypeptide to be purified is a
proteinase having a molecular weight of about 27 kDa and reduced
self-cleeavage activity relative to the self-cleavage activity of
its wild-type proteinase. In another embodiment, the polypeptide is
selected from the group consisting of tobacco etch virus (TEV) 27
kDa NIa proteinase and mutant 27 kDa NIa proteinase. In a preferred
embodiment, the polypeptide is the mutant TPSN 27 kDa NIa
proteinase, wherein the residue corresponding to Ser 219 of the
wild-type 27 kDa NIa proteinase is replaced with Asn.
[0039] Contemplated denaturing column of step (g) includes column
containing Ni-NTA resin. Contemplated methods of lysing the cells
include freeze-thaw cycles, sonication, and other enzymatic and
mechanical means. The present method also contemplates the addition
of proteinase inhibitors, preferably but not limited to PMSF,
leupeptin, and pepstatin A, to the cells before lysis. The present
invention includes the use of buffers, solubilizing agent,
renaturing agents well-known to thee skilled artisan for purifying
proteins.
[0040] Additionally, contemplated methods of using the proteinase
having a molecular weight of 27 kDa and reduced self-cleavage
activity, include cleavage of substrate. In one embodiment of using
the proteinase, the proteinase is incubated with a protein for a
sufficient amount of time to allow cleavage of the protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 shows 27 kDa NIa proteinase purification--soluble vs
insoluble preps. Samples from the soluble and insoluble
purifications of TPWT (wild-type 27 kDa NIa proteinase) and TPSN
(mutant form with Ser-Asn mutation) show that most of the 27 kDa
NIa proteinase was expressed in the insoluble fraction. Comparison
of the final products from the different purifications shows that
insoluble preparations produce greater yields with higher purity
than soluble preparations. Overloaded, renatured TPWT and TPSN
samples demonstrate sample purity as compared to a commercially
available 27 kDa NIa proteinase.
[0042] FIG. 2 shows specific activity for substrate cleavage by 27
kDa NIa proteinase. Specific activity is defined as picomoles
substrate cleaved per picomole 27 kDa NIa proteinase in 1 hour at
30.degree. C. TPWT and TPSN were compared to a commercially
available 27 kDa NIa proteinase (GibcoBRL) at several substrate
concentrations. The specific activity appears to be comparable for
all samples at the substrate concentrations tested.
[0043] FIG. 3 shows self-cleavage of 27 kDa NIa proteinase after a
5.5 week incubation. TPWT, TPSN, and a commercially available 27
kDa NIa proteinase (GibcoBRL) were incubated at various
temperatures for 5.5 weeks at a concentration of approximately
187.5 g proteinase/ml (micrograms proteinase/ml) reaction.
Comparison of the 4.degree. C. and -20.degree. C. truncated 27 kDa
NIa proteinase bands to those in the -80.degree. C. samples
indicates that self-cleavage activity is present in all samples at
4.degree. C., but is absent from the TPSN sample at -20.degree.
C.
[0044] FIGS. 4A and 4B show self-cleavage of 27 kDa NIa proteinase
at 4.degree. C. and -20.degree. C. TPWT, TPSN, and a commercially
available 27 kDa NIa proteinase (GibcoBRL) were incubated at
various temperatures for 5.5 weeks. Aliquots removed at the
timepoints shown were run on SDS-PAGE gels and quantitated using a
gel documentation system. Data were reported as the percent
intensity of the truncated 27 kDa NIa proteinase band compared to
the total intensity of both the full-length and truncated bands.
The graphs show that TPWT has comparable self-cleavage activity to
the commercially available 27 kDa NIa proteinase (assumed to have a
wild type self-cleavage site) at both 4.degree. C. and -20.degree.
C., while TPSN has reduced self-cleavage activity at 4.degree. C.
and no self-cleavage activity at -20.degree. C.
DETAILED DESECRIPTION OF THE INVENTION
A. General Description
[0045] The present invention is based on the unexpected discovery
that a single amino acid change in the internal self-cleavage site
will inhibit self-cleeavage activity of the 27 kDa NIa proteinase.
Mutating Ser at position 219 of the wild-type 27 kDa NIa proteinase
to Asn prevents the proteinase from cleaving itself.
[0046] The present invention relatese to expression,
overproduction, and purification of virus proteinase. Sources of
virus proteinase include flavi-, picorna- and potyviruses. The
present method is considered particularly suited for overproducing
potyvirus proteinases in E. coli or yeast cells.
[0047] The present invention is based on the development of a
single-column purification method for isolation of milligram
quantitites of >95% pure, active 27 kDa NIa proteinase from
inclusion body preparation. The newly developed purification method
yields about 32 mg of total pure protein from the inclusion body
preparation and greater than about 12 mg is active.
[0048] Accordingly, the present invention provides a method of
obtaining large quantitites of active 27 kDa NIa proteinase from
inclusion body preparation. The present invention also provides a
mutant 27 kDa NIa proteinase having the same substrate cleavage as
the wild-type 27 kDa NIa proteinase and a reduced self-cleavage
activity. Thus, the present invention provides a 27 kDa NIa
proteinase with increased shelf-life.
B. Definitions
[0049] Unless defined otherwise, all technical and scientific terms
used in this specification shall have the same meaning as commonly
understood by persons of ordinary skill in the art to which the
present invention pertains.
[0050] As used herein, "carrier" in a composition refers to a
diluent, adjuvant, excipient, or vehicle with which the product is
mixed.
[0051] As used herein, "carrier peptide or protein", "fusion
partner", or "heterologous protein or polypeptide" of a fusion
protein refers to the portion of the fusion protein that is added
to the protein of interest for the purpose of purification, for
stability in production, or for other reasons.
[0052] as used herein, "cocntrol sequence or element" or
"regulatory sequence or element" refers to those non-translated
regions of the vector, such as enhancers, promoters, 5' and 3'
untranslated region, which interact with host cellular proteins to
carry out transcription and translation.
[0053] As used herein, "homologs" refers to proteins having the
same or similar functions, especially proteins from different
species having the same or similar functions.
[0054] As used herein, "inclusion body" refers to distinctive
structures frequently formed in the nucleus or cytoplasm in cells
infected with certain filtrable viruses. They may be demonstrated
by means of various stains.
[0055] As used herein, "internal self-cleavage activity" or
"informal autocatalytic activity" of a protein refers to the
activity of cleaving at some site within the protein itself.
[0056] As used herein, "internal self-cleavage site" or "internal
autocatalytic site" is the site where the protein cleaves
itself.
[0057] as used herein, "isolated nucleic acid" refers to a nucleic
acid that has been separated from its naturally occurring
environment.
[0058] As used herein, "isolated polypeptide or protein" refers to
a polypeptide or protein that has been separated from its naturally
occurring environment.
[0059] As used herein, "proteinase" and "protease" are
interchangeable terms and refer to enzymes that hydrolyze (break)
polypeptide chains.
[0060] As used herein, "mutant proteinase" is a proteinase obtained
by altering the nucleic acid encoding the wild-type proteinase and
expressing the altered nucleic acid. a "mutant" is a phenotype in
which a mutation is manifested. A "mutation" is a change in the
chemistry of a nucleic acid that is perpetuated in subsequent
divisions of the cell in which it occurs.
[0061] As used herein, "specific activity" is defined as picomoles
of substrate cleaved per picomole of proteinase in one hour at
30.degree. C.
[0062] As used herein, "substrate cleavage activity" refers to the
activity of a proteinase in cleaving a specific amino acid sequence
with a specific amount of activity.
[0063] As used herein, "wild-type proteinase" is a naturally
occurring proteinase.
[0064] As used herein, "27 kDa NIa proteinase or protease" refers
to a proteinase having a molecular weight of about 27 kDa and
having the same substrate cleavage activity as the wild-type 27 kDa
NIa proteinase obtained from tobacco etch virus.
C. Specific Embodiments
1. Nucleic Acids Encoding Proteinases
[0065] The present invention provides nucleic acid molecules
encoding proteinases having the same substrate cleavage activity as
the wild-type 27 kDa NIa proteinase, and preferably the nucleic
acid molecules are in isolated form. In one embodiment, nucleic
acid molecules provided by the present invention encode a
proteinase having 27 kDa and reduced self-cleavage activity as
compared to the self-cleavage activity of its wild-type proteinase.
In another embodiment, nucleic acid molecules of the present
invention encode a mutant form of the 27 kDa NIa proteinase
comprising an amino acid substitution corresponding to position 219
of its wild-type proteinase, prefeerably Ser substituted with Asn,
having the same substrate cleavage activity as the wild-type
proteinase and having reduced self-cleavage activity relative to
its wild-type proteinase.
[0066] The nucleic acid molecules of the invention include
deoxyribonucleic acids (DNAs), both single- and double-stranded
deoxyribonucleic acids. However, they can also be ribonucleic acids
(RNAs), as well as hybrid RNA:DNA double-stranded molecules.
Contemplated nucleic acid molecules also include genomic DNA, cDNA,
mRNA, and antisense molecules. The nucleic acids molecules of the
present invention also include native or synthetic, RNA, DNA, or
cDNA, that encode a proteinase protein, or the complementary strand
thereof, including but not limited to nucleic acid found in a
proteinase expressing organism, such as the tobacco etch virus.
[0067] The nucleic acid sequence encoding the proteinase can be,
for instance, substantially or full synthetic. See, for example,
Goeddel et al., Proc. Natl. Acad. Sci. USA, 76, 106-110, 1979. For
recombinant expression purposes, codon usage preferences for the
organism in which such a nucleic acid is to be expressed are
addvantageously considered in designing a synthetic
proteinase-encoding nucleic acid. Codon usage preferences for
different organisms are well known to the skilled artisan. Since
the nucleic acid code is degenerate, numerous nucleic acid
sequences can be used to create the same amino acid sequence.
[0068] The nucleic acid molecules of the present invention can
encode proteinases having the same substrate cleavage activity as a
wild-type 27 kDa NIa proteinase. For example, the nucleic acid
molecules can encode a proteinase having 27 kDa and reduced
self-cleavage activity or a mutant form of the 27 kDa NIa
proteinase having the same substrate cleavage activity and having
reduced self-cleavage activity as compared with it wild-type
proteinase, piconaviral 3C proteinases, cellular serine proteinases
such as chymotrypsin trypsin proteinases, proteinases that have
internal self-cleavage sites similar to the internal self-cleavage
site of the 27 kDa NIa proteinase, mutant forms of such proteinases
with reduced self-cleavage activity as compared to its wild-type
proteinase, and proteinases that recognize the same extended seven
amino acid sequence (E-X-X-Y-X-Q.dwnarw.S/G) (SEQ ID NO: 1)
substrate cleavage site as the 27 kDa NIa proteinase.
[0069] In one embodiment, the nucleic acids of the present
invention encode a proteinase having 27 kDa and reduced
self-cleavage activity as compared to its wild-type proteinase. In
another embodiment, the nucleic acids of the present invention
encode a mutant form of the 27 kDa NIa proteinase comprising an
amino acid sequence in which the residue corresponding to 219 of
the 27 kDa NIa proteinase is replaced with another residue,
preferably Asn.
[0070] Nucleic Acid Molecules Encoding Mutant forms and Allelic
Forms of Proteinases. To construct mutant forms of proteinases
having the same substrate cleavage activity as the wild-type 27 kDa
NIa proteinase and having reduced self-cleavage activity, the
nucleic acid encoding the wild-type proteinase can be used as a
starting point and modified to form the desired mutants. For
example, in the preferred embodiment, the nucleic acid sequence
encoding the wild-type 27 kDa NIa proteinase is mutated such that
the Ser corresponding to positiion 219 in the encoded amino acid
sequence is replaced with another amino acid, preferably Asn.
[0071] Further, with an altered amino acid sequence, numerous
methods are known to delete sequence from or mutate nucleic acid
sequences that encode a polypeptide and to confirm the function of
the polypeptides encoded by these deleted or mutated sequences.
Accordingly, the invention also relates to a mutated or deleted
version of a proteinase nucleic acid that encodes a proteeinase
that has the same substrate cleavage activity as the wild-type 27
kDa NIa proteinase.
[0072] Conservative variants of the wild-type 27 kDa NIa
proteinases or its naturally occurring isoforms and homologs are
encompassed by the present invention. Such conservative mutations
include mutations that switch one amino acid for another within one
of the following groups: [0073] 1. Small aliphatic, nonpolar or
slightly polar residues: Ala, Ser, Thr, Pro and Gly; [0074] 2.
Polar, negatively charged residues and their amides: Asp, Asn, Glu
and Gln; [0075] 3. Polar, positively charged residues: His, Arg and
Lys; [0076] 4. Large aliphatic, nonpolar residues: Met, Leu, Ile,
Val and Cys; and [0077] 5. Aromatic residues: Phe, Tyr and Trp. The
types of substitutions selected may be based on the analysis of the
frequencies of amino acid substitutions between homologous proteins
of different species developed by Schulz et al., Principles of
Protein Structure, Springer-Verlag, 1978, pp. 14-16, on the
analysis of structure-forming potentials developed ty Chou and
Fasman, Biochemistry 13, 211, 1974 or other such methods reviewed
by Schulz et al., Principles in Protein Structure, Springer-Verlag,
1978, pp 108-130, and on the analysis of hydrophobicity patterns in
proteins developed by Kyte and Doolittle, J. Mol. Biol. 157:
105-132, 1982.
[0078] The present invention also contemplates nucleic acids
encoding naturally occurring allelic variants of the proteinases
described above. In a preferred embodiment, allelic variants even
though possessing a slightly different amino acid sequence than the
naturally occurring wild-type 27 kDa proteinase will have the
requisite ability to recognize and cleave the heptapeptide sequence
E-X-X-Y-X-Q.dwnarw.S/G (SEQ ID NO: 1). The present invention also
contemplates conservative variants that do not affect the ability
of the proteinase to recognize and cleave the heptapeptide sequence
E-X-X-Y-X-Q.dwnarw.S/G (SEQ ID NO: 1). The present invention
includes 27 kDa NIa proteinase with altered overall charge,
structure, hydrophobic/hydrophilic properties by amino acid
substitutions, insertions, or deletions but still possess the
ability to recognize and cleave the heptapetide.
[0079] Preferably, the nucleic acids will encode proteinases having
the same substrate cleavage activity as the wild-type 27 kDa NIa
proteinase and having at least about 70% sequence identity, more
preferably, at least about 80% sequence identity, even more
preferably, at least about 85% sequence identity, yet more
preferably at least about 90% sequence identity, and most
preferably at least about 95% sequence identity to a wild-type 27
kDa NIa proteinase or other naturally occurring isoforms having the
same substrate cleavage activity.
[0080] Numerous methods for determining percent homology are known
in the art. One preferred method is to use version 6.0 of the GAP
computer program for making sequence comparisons. The program is
available from the University of Wisconsin Genetics Computer Group
and utilizes the alignment method of Needleman and Wunsch, J. Mol.
Biol. 48, 443, 1970, as revised by Smith and Waterman Adv. Appl.
Math. 2, 482, 1981. Numerous methods for determining percent
identity are also known in the art, and a preferred method is to
the FASTA computer program, which is also available from the
Univeersity of Wisconsin Genetics Computer Group.
[0081] Additionally, the invention includes substantially pure
nucleic acids that hybridize under stringent conditions to a
nucleic acid encoding a proteinase having the same substrate
cleavage activity as the wild-type 27 kDa NIa proteinase. Stringent
hybridization conditions are conditions in which hybridization to a
labeled known nucleic acid sequence yields a clear and detectable
squence. Stringent conditions are those that (1) employ low ionic
strength and high temperature for washing, for example, with 0.015
M NaCl, 0.0015 M sodium titrate, 0.1% SDS at 50.degree. C.; (2)
employ during hybridization a denaturing agent such as formamide,
for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium nitrate
at 42.degree. C.; and (3) employ 50% formamide, 5.times. SSC (0.75
M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),
0.1% sodium pyrophosphate, 5.times. Denhardt's solution, sonicated
salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate
at 42.degree. C., followed with washes at 42.degree. C in
0.2.times. SSC and 0.1% SDS. A skilled artisan can readily
determine and vary the stringency conditions appropriately to
obtain a clear and detectable hybridization signal.
[0082] Nucleic Acid Molecules Encoding Fusion Proteins. The nucleic
acid molecules of the present invention also encode fusion proteins
comprising a proteinase such as those described in the previous
paragraphs fused to a heterologous protein or polypeptide. In one
embodiment, the fusion proteins of the present invention comprise
the proteinase having 27 kDa and reduced self-cleavage activity as
compared to its wild-type proteinase fused to a heterologous
protein. In another embodiment, the fusion protein comprises the
mutant 27 kDa NIa proteinase comprising an amino acid sequence in
which the residue corresponding to Ser 219 of the 27 kDa NIa
proteinase is replaced with another residue, preferably Asn, fused
to a heterologous protein.
[0083] Nucleic acids encoding various heterologous proteins may be
fused to the nucleic acid encoding the proteinase. For example,
nucleic acid encoding affinity tags such as the His tags,
antibodies, or carrier peptides or proteins having specific binding
properties such as Staphylococcus aureus protein A and the
carbohydrate recognition domain (CRD) of galactose-specific rat
hepatic lectin (Taylor and Drickamer, 1991), may be fused to the
proteinase for purification of the proteinase by affinity
chromatography. Nucleic acid encoding substrate cleavage sites of
thrombin or factor X may be fused to the nucleic acid encoding the
proteinase for ease of removal of carrier peptide. Additionally,
fusion proteins tend to be more soluble than a single protein,
contributing to higher yields and simpler purification. The fusion
partner for the proteinase may be selected on the basis of
transport characteristics to assure that the fusion protein is
secreted into either the periplasmic space or the growth medium.
Moreover, the fusion partner may also be selected for increasing
the stability of the proteinase in the preferred expression system
for obtaining large quantities of the protein. For example, fusion
proteins are usually more stable in bacteria than the native
eukaryotic proteinase.
[0084] Nucleic Acid Encoding Proteins or Polypeptides Expressed in
Inclusion Bodies. Nucleic acids encoding proteins, polypeptides, or
fusion proteins or polypeptides that form inclusion bodies in cells
are also encompassed by the present application. Such nucleic acids
can be expressed in host cells to produce large quantities of the
proteins or polypeptides in inclusion bodies, as discussed below.
The proteins or polypeptides are then purified by the purification
method provided by the present invention, also discussed below.
2. Polypeptides or Proteins
[0085] Polypeptides of the invention include all proteinases having
the same substrate cleavage activity as the wild-type 27 kDa NIa
proteinase, and preferably in isolated or purified form. The
present invention also includes these proteinases in native or
synthetic form, including but not limited to polypeptides purified
from a proteinase-expressing organism. In one embodiment, the
polypeptides of the present invention comprise a proteinase having
27 kDa and reduced self-cleavage activity as compared to its
wild-type proteinase. In another embodiment, the polypeptides of
the present invention comprise a mutant form of the wild-type 27
kDa NIa proteinase having the same substrate cleavage activity as
the wild-type proteinase, but with reduced self-cleavage activity
as compared to its wild-type proteinase, and having amino acid
corresponding to position 219 of the wild-type NIa proteinase
substituted with another amino acid, preferably Asn.
[0086] Structurally, the 27 kDa NIa proteinase has been reported to
be similar to the trypsin-like family of cellular serine
proteinases, such as chymotrypsin or trypsin, with the substitution
of Cys for serine as the active site nucleophile (Blazan et al.,
1990; Dougherty et al., 1989). Dougherty et al. (1989) discloses
the catalytic triad of 27 kDa NIa proteinase to be composed of His,
Asp, and Cys which is similar to the catalytic triad found in other
viral proteinases (Dougherty et al., 1989). However, unlike the
other proteinases, the 27 kDa proteinase recognizes and extended
heptapeptide sequence, E-X-X-Y-X-Q.dwnarw.S/G (positions
P6-P1.dwnarw.P40 1; X is an amino acid) (SEQ ID NO: 1), and cleaves
within the heptapeptide sequence (Dougherty et al. 1989a; Dougherty
et al. 1988; Dougherty et al., 1989b).
[0087] Proteinases having the same substrate cleavage activity as a
wild-type 27 kDa NIa proteinase include but are not limited to
polypeptides comprising the wild-type 27 kDa NIa proteinase or a
mutant form thereof having the same substrate cleavage activity and
having reduced self-cleavage activity as compared with its
wild-type proteinase, piconaviral 3C proteinases, cellular serine
proteinases such as chymotrypsin trypsin proteinases, proteinases
that have internal self-cleavage sites similar to the internal
self-cleavage site of the 27 kDa NIa proteinase, mutant forms of
such proteinases with reduced self-cleavage activity as compared to
its wild-type proteinases, and proteinases that recognize the same
extended seven amino acid sequence (E-X-X-Y-X-Q.dwnarw.S/G, SEQ ID
NO: 1) substrate cleavage site as the 27 kDa NIa proteinase.
[0088] In one embodiment, polypeptides of the present invention
comprise a proteinase having a molecular weight of 27 kDa and
reduced self-cleavage activity as compared to the wild-type 27 kDa
NIa proteinase. In another embodiment, the polypeptides of the
present invention comprise the mutant form of the 27 kDa NIa
proteinase having an amino acid sequence in which the residue
corresponding to Ser219 of the 27 kDa NIa proteinase is replaced
with another residue, preferably Asn.
[0089] Mutant Forms and Allelic Forms of Proteinases. The present
invention also include mutant forms of proteinases having the same
substrate cleavage acitity as the wild-type 27 kDa NIa proteinase
and having reduced self-cleavage activity. As discussed above, to
construct mutant forms of proteinases, the nucleic acid encoding
the wild-type proteinase can be used as a starting point and
modified to form the desired mutants. For example, in the preferred
embodiment, the nucleic acid sequence encoding the wild-type 27 kDa
NIa proteinase is mutated such that Ser corresponding to position
219 in the encoded amino acid sequence is replaced with another
amino acid, preferably Asn. Further, numerous methods are known to
delete and mutate nucleic acid sequences that encode a polypeptide
and to confirm the function of the polypeptides encoded by these
deleted or mutated sequences. Accordingly, the invention also
provides mutated or deleted version of a proteinase that has the
same substrate cleavage activity as the wild-type 27 kDa NIa
proteinase.
[0090] Conservative variants of the wild-type 27 kDn NIa
proteinases or its naturally occurring isoforms and homologs are
encompassed. Such conservative mutations have been discussed under
the previous section. The present invention also contemplates
conservative variants that do not affect the ability of the
proteinase to recognize and cleave the heptapeptide sequence
E-X-X-Y-X-Q.dwnarw.S/G (SEQ ID NO: 1). The present invention
includes 27 kDa NIa proteinase with altered overall charge,
structure, hydrophobic/hydrophilic properties by amino acid
substitutions, insertions, or deletions but still possess the
ability to recognize and cleave the heptapeptide.
[0091] The present invention also contemplates naturally occurring
allelic variants of the proteinases having the same substrate
cleavage activity as the 27 kDa NIa proteinase. In a preferred
embodiment, allelic variants even though possessing a slightly
different amino acid sequence than the naturally occurring
wild-type 27 kDa proteinase will have the requisite ability to
recognize and cleave the heptapeptide sequence
E-X-X-Y-X-Q.dwnarw.S/G (SEQ ID NO: 1).
[0092] Preferably, proteinases having the same substrate cleavage
activity as the wild-type 27 kDa NIa proteinase and at least about
70% sequence identity, more preferably, at least about 80% sequence
identity, even more preforably, at least about 85% sequence
identity, yet more preferably at least about 90% sequence identity,
and most preferably at least about 95% sequence identity to a
wild-type 27 kDa NIa proteinase or other naturally occurring
isoforms having the same substrate cleavage activity.
[0093] Numerous methods for determining percent homology are known
in the art. One preferred method is to use version 6.0 of the GAP
computer program for making sequence comparisons. The program is
available from the University of Wisconsin Genetics Computer Group
and utilizes the alignment method of Needleman and Wunsch, J. Mol.
Biol. 48 443, 1970, as revised by Smith and Waterman Adv. Appl.
Math. 2, 482, 1981. Numerous methods for determining percent
identity are also known in the art, and a preferred method is to
use the FASTA computer program, which is also available from the
University of Wisconsin Genetics Computer Group.
[0094] Fusion Proteins. The present invention also provides fusion
proteins comprising a proteinase having the same substrate cleavage
activity as the 27 kDa NIa proteinase fused to a heterologous
protein or polypeptide. In one embodiment, the fusion proteins of
the present invention comprise a proteinase having a molecular
weight of about 27 kDa and reduced self-cleavage activity as
compared to its wild-type proteinase fused to a heterologous
protein. In another embodiment, the fusion proteins of the present
invention comprise a mutant 27 kDa NIa proteinase having an amino
acid sequence in which the residue corresponding to Ser219 of the
27 kDa NIa proteinase is replaced with another residue, preferably
Asn, fused to a heterologous protein.
[0095] As discussed earlier, various heterologous proteins may be
fused to the proteinase of the present invention (see below
also).
[0096] Compositions. The present invention also provides
compositions comprising an isolated proteinase having the same
substrate cleavage activity as the wild-type 27 kDa NIa proteinase
and a carrier. The composition may comprise a dry formulation or an
aqueous solution. The carrier may be any compound that does not
affect the substrate cleavage activity of the proteinase. Carrier
could be a diluent, an excipient, or even a stabilizer. A specific
example of a carrier could be buffer or water, which does not
affect the stability of the proteinase.
[0097] Uses of Proteinases with Identical Substrate Cleavage
Activity as the 27 kDa NIa Proteinase. The present invention also
provides methods of using proteinases with the same substrate
cleavage activity as the 27 kDa NIa proteinase. Proteinases of the
present invention can be used to cleave polypeptides comprising the
heptapeptide sequence B-X-X-Y-X-Q.dwnarw.S/G (SEQ ID NO; 1). The
mutant forms of the 27 kDa NIa proteinase with decreased self
cleavage actitiy are more stable than the wild-type proteinase and
have a longer shelf-life.
[0098] As discussed earlier, Parks et al. (1994) and Johnson et
al., U.S. Pat. No. 5,532,142, disclose the use of the 27 kDa NIa
proteinase as a tool for purifying and obtaining large quantities
of desired proteins. As shown by Parks et al. and Johnson et al.,
to obtain large quantities of a desired protein, the protein is
fused to a carrier protein and a substrate cleavage site recognized
by the 27 kDa NIa proteinase is inserted between the two proteins.
The 27 kDa NIa proteinase is selected for separating the carrier
protein from the desired protein because the 27 kDa NIa proteinase
exhibits unique characteristics. Unlike other proteinases, the 27
kDa proteinase exhibits high specificity, insensitivity to many
proteinase inhibitors used in protein purification, and efficient
cleavage under broad range of temperatures (Polayes et al., 1994).
Moreover, the protein of interest can be easily separated from the
carrier peptide and the 27 kDa proteinase.
[0099] The present invention provides mutant forms of the 27 kDa
NIa proteinase with the same substrate activity as the wild-type
proteinase and with decreased self-cleavage activity. The mutant 27
kDa NIa proteinase of the present invention are also useful as
tools for purifying and obtaining large quantities of desired
proteins.
[0100] Proteins or Polypeptides that Form Inclusion Bodies.
Proteins, polypeptides, fusion proteins or polypeptides that form
inclusion bodies in cells are also encompassed by the present
application. Such proteins, polypeptides, or fusion proteins,
either produced by recombinant means or present in their native
source, are then purified by the purification method provided by
the present invention, discussed below.
[0101] In a preferred embodiment, the fusion proteins comprising a
protein of interest is fused to a carrier protein or fusion partner
that facilitates its isolation. Examples of carrier proteins are
not limited to any particular protein, but may be selected from a
wide variety of proteins such as beta galactosidase, ubiquitin,
glutathione S-transferase, alkaline phosphatase, maltose binding
protein, Protein A, polyhistidines, monoclonal antibody epitopes
and so forth. Carrier proteins typically will be selected on the
basis of characteristics contributing to easy isolation, most
desirable being those that are readily secreted by the
microorganisms or which have some property or feature which
facilitates isolation and purification of the protein. Glutathione
S-transferase, maltose binding protein and polyhistidine sequences,
for example, are generally preferred because there are readily
available affinity columns to which they can be bound and eluted.
Other suitable fusion partners include antigenic tags that reaily
bind to corresponding antibodies or proteins that have special
affinity properties, for example, selective binding to particular
metals, as with polyhistidine peptide binding to nickel.
3. Recombinant Production of Proteinases
[0102] Vectors and Expression vectors. The present invention
provides vectors and expression vectors comprising a nucleic acid
encoding a proteinase having the same substrate cleavage activity
as the wild-type 27 kDa NIa proteinase. In a preferred embodiment,
the vectors or expression vectors comprise a nucleic acid encoding
a proteinase having a molecular weight of about 27 kDa and reduced
self-cleavage activity as compared to its wild-type proteinase. In
a more preferred embodiment, the vectors or expression vectors
comprise the nucleic acid encoding a mutant form of the 27 kDa NIa
proteinase having the same substrate cleavage activity as its
wild-type 27 kDa NIa proteinase and reduced self-cleavage activity
as compared to its wild-type proteinase.
[0103] The present invention also provides vectors and expression
vectors containing the nucleic acids encoding fusion proteins and
encoding any protein that forms inclusion bodies in cells.
Preferably, the fusion proteins comprise a proteinase having the
same substrate cleavage activity as the 27 kDa NIa proteinase and a
heterologous protein. More preferably, the proteinase of the fusion
protein is a mutant form of 27 kDa NIa proteinase having reduced
self-cleavage activity and has an amino acid sequence in which the
residue corresponding to position 240 is replaced with another
amino acid, preferably Asn.
[0104] Vectors or cassettes useful for the transformation and
transfection of suitable host cells are well known in the art.
Typically, the vectors or cassettes contain sequences directing
transcription and/or translation of the relevant gene, a selectable
marker, and sequences allowing antonomous replication or
chromosomal integration. In an autonomously replicating vectors,
i.e., a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, and extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon may
be used.
[0105] Suitable vectors for the present invention comprise a region
5' of the gene which harbors transcriptional initiation regulation
or control and a region 3' of the DNA fragment which regulates
transcriptional termination. It is most preferred when both
regulatory regions are derived from nucleic acids homologous to the
transformed host cell, although it is to be understood that such
regulatory regions need not be derived from the nucleic acis native
to the specific species chosen as a production host.
[0106] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters such as the hybride lacZ promotoer of the BLUESCRIPT
phagemid (Stratagene, LaJolla, Calif.) or PSPORTl plasmid (Gibco
BRL) and the like may be used. The baculovirus polyhedrin promoter
may be used in insect cells. Promoters or enhancers derived from
the genomes of plant cells (e.g., heat shock, RUBISCO; and storage
protein genes) or from plant viruses (e.g., viral promoters or
leader sequences) may be cloned into the vector. In mammalian cell
systems, promoters from mammalian genes or from mammalian viruses
are preferable. It it is necessary to generate a cell line that
contains multiple copies of the sequence encoding the proteinase or
protein formed in inclusion bodies, vectors based on SV40 or EBV
may be used with an appropriate selectable marker.
[0107] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for the proteinase or
protein that forms inclusion bodies in a cell. For example, when
large quantities of the protein are needed for the induction of
antibodies or for use as a tool in the purification of proteins,
vectors which direct high level expression of fusion proteins that
are readily purified may be used. Such vectors include, but are not
limited to, the multifunctional E. coli cloning and expression
vectors such as BLUESCRIPT (Stratagene), in which the sequence
encoding the proteinase may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced. An example of such a vector includ pIN vectors (Van
Hecke, G. and S.M. Schuster (1989) J. Biol. Chem.
264:5503-5509).
[0108] pGEX vectors (Promega, Madison, Wis.) are used to express
foreign polypeptides as fusion proteins with a heterologous protein
such as glutathione S-transferase (GST). In general, fusion
proteins are soluble and can easily be purified from lysed cells by
adsorption to glutathione-agarose beads followed by elution in the
presence of free glutathione. Proteins made in such systems may be
designed to include a protease cleavage site, such as the substrate
cleavage site of the 27 kDa NIa proteinase, so that the purified
polypeptide of interest can be easily released from the GST moiety.
The fusion proteins may also comprise a preferred proteinase or any
protein that forms inclusion bodies in a cell and a carrier peptide
or ptotein, such as the His tag, for affinity purification of the
proteinase or protein. A further discussion of vectors which
contain fusion proteins is provided in Kroll, D. J. et al. (1993;
DNA cell Biol. 12:441-453).
[0109] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing consitutive or inducible promoters such as alpha factor,
alcohol oxidase, and PgH may be used (Grant et al. (1987) Methods
Enzymol. 153:516-544). In cases where plant expression vectors are
used, the expression of sequences encoding the proteinase or
protein that forms inclusion bodies in a cell may be driven by any
of a number of promoters. For example, viral promoters such as the
35S and 19S promoters of CAMV may be used alone or in combination
with the omega leader sequence from TMV (Takamaisu, N. (1987) EMBO
J. 6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probi.
Cell Differ, 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Murry, L. E. in
McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill,
New York, N.Y.; pp. 191-196).
[0110] An insect system may also be used to express the proteinase
or protein that forms inclusion bodies in a cell. For example, in
one such system, Autographa californica nuclear polyhedrosis virus
(AcNPV) is used as a vector to express foreign genes in Spodoptera
frugiperda cells or in Trichoplasia larvae. The sequences encoding
the protein may be cloned into a non-essential region of the virus,
such as the polyhedrin gene, and placed under control of the
polyhedrin promoter. Successful insertion of the protein will
render the polyhedrin gene inactive and produce recombinant virus
lacking coat protein. The recombinant viruses may then be used to
infect, for example, S. frugiperda cells or Trichoplusia larvae in
which PLBP may be expressed (Engelhard, E. K. et al. (1994) Proc.
Nat. Acad. Sci. 91:3224-3227).
[0111] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding the proteinase or protein
that form inclusion bodies in a cell may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain a viable virus which is capable of expressing PLBP in
infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl.
Acad. Sci. 81:3655-3659). In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0112] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding the proteinase or
protein formed in inclusion bodies. Such signals include the ATG
initiation codon and adjacent sequences. In cases where sequences
encoding a protein, its initiation codon, and upstream sequences
are inserted into the appropriate expression vector, no additional
transcriptional or translational regulatory signals may be needed.
However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous tranlational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. the efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0113] Host Cells. The present invention also provides host cells,
comprising a nucleic acid sequence encoding a proteinase as
described above or a protein that forms inclusion bodies in a cell,
which are used in the recombinant production of the encoding the
proteinase or protein. A vector comprising the nucleic acid
sequence of the present invention is introduced into a host cell so
that the vector is maintained as a chomosomal integrant or as a
self-replicating extra-chromosomal vector as described earlier. The
choice of a host cell will to a large extent depend upon the gene
encoding the polypeptide and its source. The host cell may be
unicellular microorganism, e.g., a prokaryote, or a non-unicellular
microorganism, e.g., a eukaryote. The host cell may be a eukaryote
selected from the group consisting of mammalian cell, insect cell,
plant cell or fungal cell.
[0114] Useful unicellular cells are bacterial cells such as gram
positive bacteria including, but not limited to, a Bacillus cell,
e.g., Bacillus alkalophilus, Bacillus amyloliquefacions, Bacillus
brevis, Bacillus circulans, Bacillus glansii, Bacillus coagulans,
Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus stearothermophilus, Bacillus subtilis, and
Bacillus thuringiensis; or a Streptomyces cell, e.g, Streptomyces
lividans or Streptomyces murinus, or gram negative bacteria such as
E. coli and Pseudomonas sp. In a preferred embodiment, the
bacterial host cell is E. coli. Various strains of E. coli (e.g.,
HB101, DH5, DH10, and MC1061) are well-known as host cells in the
field of biotechnology.
[0115] Mammalian cells, such as Chinese hamster ovary cells (CHO)
or 3T3 cells may be used in the present invention. the selection of
suitable mammalian host cells and methods for transformation,
culture, amplification, screening and product production and
purification are known in the art. Other suitable mammalian cell
lines, are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell
line. Further exemplary mammalian host cells include primate cell
lines and rodent cell lines, including transformed cell lines.
Normal diploid cells, cell strains derived from in vitro culture of
primary tissue, as well as primary explants, are also suitable.
Candidate cells may be genotypically deficient in the selection
gene, or may contain a dominantly acting selection gene. Other
suitable mammalian cell lines include but are not limited to, HeLa,
mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH
mice, BHK or HaK hamster cell lines.
[0116] Many strains or yeast cells known to those skilled in the
art are also available as host cells for expression of the
polypeptides of the present invention. Additionally, where desired,
insect cells may be utilized as host cells in the method of the
present invention (Miller et al., 1986 Genetic Engineering
8:277-298).
[0117] The introduction of a vector into a bacterial host cell may,
for instance, be effected by protoplast transformation (see, e.g.,
Chang and Cohen, 1979, Molecular General Genetics 168: 111-115),
using competent cells (see, e.g., Young and Spizizin, 1961, Journal
of Bacteriology 81: 823-829, or Dubnan and Davidoff-Abelson, 1971,
Journal of Molecular Biology 56: 209-221), electroporation (see,
e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or
conjugation (see, e.g., Koehler and Thorne, 1987, Journal of
Bacteriology 169: 5771-5278).
[0118] Host cells transformed with nucleotide sequences encoding
proteinase and protein that form inclusion bodies may be cultured
under conditions suitable for the expression and recovery of the
proteinase from cell culture. The protein produced by a transformed
cell may be secreted, contained intracellularly, or contained with
the inclusion factor depending on the sequence and/or the vectory
used. As will be understood by those of skill in the art,
expression vectors containing polynucleotides which encode the
protein may be designed to contain signal sequences which direct
secretion of the protein through a prokaryotic or eukaryotic cell
membrane.
[0119] Other constructions may be used to join sequences encoding
the protein to nucleotide sequence encoding a polypeptide domain
which will facilitate purification of soluble proteins. Such
purification facilitating domains include, but are not limited to,
metal chelating peptides such as histidine-tryptophan modules that
allow purification on immobilized metals, protein A domains that
allow purification on immobilized immunoglobulin, and the domain
utilized in the FLAGS extension/affinity purification system
(Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker
sequences such as those specific for Factor XA or enterokinase
(Invitrogen, San Diego, Calif.) or the 27 kDa NIa proteinase
cleavage site between the purification domain and the protein may
be used to facilitate purification. One such expression vector
provides for expression of a fusion protein containing the protein
and a nucleic acid encoding 6 histidine residues preceding a
thioredoxin or an enterokinase cleavage site. The histidine
residues facilitate purification on IMAC (immobilized metal ion
affinity chromatography) as described in Porath, J. et al. (1992,
Prot. Exp. Purif. 3: 263-281) while the enterokinase cleavage site
provides a means for purifying from the fusion protein.
[0120] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and WI38), are available from the American
Type Culture Collection (ATCC; 10801 University Boulevard,
Manassas, Va., 20110-2209) and may be chosen to ensure the correct
modification and processing of the foreign protein.
[0121] For long-term, high-yield production of proteinases or
proteins that form inclusion bodies in a cell, stable expresssion
is preferred. For example, cell lines which stably express
proteinase or proteins that form inclusion bodies may be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may be allowewd to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells which successfully express the introduced
sequences. Resistant clones of stabley transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0122] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al. (1977)
Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy, L et
al. (1980) Cell 22:817-23) genes which can be employed in `or aprt`
cells, respectively. Also, antimetabolite, antibiotic or herbicide
resistance can be used as the basis for selection; for example,
dhfr which confers resistance to methotrexate (Wigler, M. et al.
(1980) Proc. Natl. Acad. Ssci, 77:3567-70); npt, which confers
resistance to the aminoglycosides, neomycin and g-418
(Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14); and als
or pat, which confers resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci
85:8047-51). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, -glucuronidase and
its substrate GUS, and luciferase and its substrate luciferin,
being widely used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributtable to a specific vector system (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131).
[0123] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. for example, if the
sequence encoding a protein ase or a protein thaty forms inclusion
bodies in a cell, is inserted within a marker gene sequence,
transformed cells containing sequences encoding the protein can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in transem with a sequence encoding
protein under the control of a single promoter. Expression of the
marker gene in response to induction or selection usually indicates
expression of the tandem gene as well Alternatively, host cells
which contain the nucleic acid sequence encoding the protein and
express the protein may be identified by a variety of procedures
known to those of skill in the art. These procedures include, but
are not limited to, dNA-DNA or DNA-RNA hybridizations and substrate
cleavage assay or ummunoassay techniques which include membrane,
solution, or chip based technologies for the detection and/or
quantification of nucleic acid or protein.
[0124] Production of Protein from Host Cells. The present invention
also provides methods for producing a proteinase or a protein that
forms inclusion body in a cell comprising (a) cultivating the host
cell under conditions that allow expression of the protein; and (b)
recovering the protein. The proteinase of the present invention has
a substrate cleavage activity that is identical to that of the 27
kDa NIa proteinase. Preferably, the proteinase has a molecular
weight of about 27 kDa and a reduced self-cleavage activity as
compared to its wild-type proteinase. More preferably, the
proteinase is a mutant form of the 27 kDa NIa proteinase, and
comprises an amino acid sequence in which the residue corresponding
to position 219 of the wild-type 27 kDa NIa is replaced with
another amino acid, preferably Asn.
[0125] In the production methods of the present invention, the
cells are cultivated in a nutrient medium suitable for production
of the proteins of the present invention using methods known in the
art. For example, the cell may be cultivated by shake-flask
cultivation, small-scale or large-scale fermentation (including
continuous, batch, fed-batch, or solid state fermentations) in
laboratory or industrial fermenters performed in a suitable medium
and under conditions allowing the polypeptide to be expressed
and/or isolated. The cultivation takes place in a suitable nutrient
medium comprising carbon and nitrogen sources and inorganic salts,
using procedures known in the art. Suitable media are available
from commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection). If the protein is secreted into the nutrient medium,
the polypeptide can be recovered directly from the medium. If the
protein is not secreted, it can be recovered from cell lysates as
described below.
[0126] The proteins may be detected using methods known in the art
that are specific for the polypeptides. These detection methods may
include use of spcific antibodies, formation of an enzyme product,
or disappearance of an enzyme substrate. For example, an enzyme
assay may be used to determine the activity of the protein.
[0127] The resulting protein may be recovered by methods described
below.
4. Purification of Proteins
[0128] General Procedure for Purification of Proteins. The proteins
of the present invention produced from host cells may be recovered
by methods known in the art. For example, the polypeptide may be
recovered from the nutrient medium by conventional procedures
including, but not limited to, centrifugation, filtration,
extraction, spray-drying, evaporation, or precipitation.
[0129] The proteins of the present invention may be purified by a
variety of procedures known in the art including, but not limited
to, chromatography (e.g., ion exchange, affinity, hydrophobic,
chromatofocusing, and size exclusion), electrophoretic procedures
(e.g., preparative isoelectric focusing), differential solubility
(e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction
(see, e.g., Protein Purification, J. -C Janson and Lars Ryden,
editors, VCH Publishers, New York, 1989).
[0130] In one embodiment of the invention, when the protein is
secreted into the culture medium, the step of collecting the
conditioned culture medium is followed by the step of purifying the
protein. the protein in the collected medium may be purified by any
method known in the art, such as selective precipitation, affinity
chromatography, dialysis, immunoprecipitation, ion-exchange
chromatography, size-exclusion chromatography, hydrophobic
interaction chromatography, or reversed-phase chromatography (Guide
to Protein Purification, in Methods in Enzymology 182 (Murray P.
Deutscher ed., 1990), Remington: The Science and Practice of
Pharmacy 534-58 (Alfonso R. Gennaro ed., 19th ed. 1995)).
Chromatography can be carried out using conventional columns or by
HPLC (high performance liquid chromatography) or FPLC (fast protein
liquid chromatography).
[0131] In another embodiment of the invention, the protein is
purified by anion-exchange chromatography. Methods of purifying
proteins using anion-exchange chromatography are well known to
those skilled in the art (See, e.g., Edward R. Rossomando,
Ion-Exchange Chromatography, in Guide to Protein Purification,
Methods of Enzymology 182, 309-16 (Murray P. Deutscher ed., 1990)).
In an alternate embodiment of the invention, the protein is
purified by affinity chromatography over a protamine-agarose
column, such as a protamine-Sepharose.RTM. (Pharmacia-LKB) column.
Methods of purifying the proteins of the present invention using
protamine-agarose columns are known to those skilled in the art
(Dempfle and Heene, Thromb. Res. 46, 19 (1987)). Preferably, the
proteins of the present invention are purified by affinity
chromatography using a column containing Ni-NTA Superflow resin
(nickel-nitilotriacetic acid-agarose, Qiagen).
[0132] In an alternate embodiment of the invention, the protein is
purifed by immunoaffinity chromatography using polyclonal or
monoclonal antibodies raised against the intact protein or peptides
thereof of the present invention. Methods of producing and using
polyclonal and monoclonal antibodies are well known in the art (Ed
Harlow & David Lane, Antibodies: A Laboratory Manual (1988);
Norman A. Staines, Monoclonal Antibodies, in Biochemical Research
Techniques: A Practical Introduction (John M. Wrigglesworth Ed.,
1983)). Likewise, methods of performing affinity chromatography
using polyclonal and monoclonal antibodies are also well knwon in
the art. (Ed Harlow & David Lane, Antibodies: A Laboratory
Manual (1988); Steven Ostrove, Affinity Chromatography. General
Methods, in Guide to Protein Purification, Methods of Enzymology
182, 357-71 (Murray P. Deutscher ed., 1990)).
[0133] In another embodiment of the invention, when the prtein is
not secreted into the culture medium, it is necessary to remove the
protein from the cell by lysing the cells using methods well known
in the art such as sonication or freeze-thawing, followed by
isolating the protein from the cell extract. The cell extract may
be prepared or incubated in the presence of cell protease
inhibitors. The plant virus cleavage site is not susceptible to
proteolysis by ordinary cell proteases. The plant virus proteinase
itself is unaffected by ordinary protease inhibitors so that such
inhibitors may be added in amounts sufficient to inactivate cell
proteases. Typical and commonly used cell protease inhibitors
include leupeptin, pepstatin A, PMSF, E-64, TLCK, bestatin and
aprotinin. However, any of a number of proteinase inhibitors may be
employed so long as they are not inhibitors of the proteinase used
to release a foreign protein from the carrier protein. The
practitioner will typically culture in a media or grow in an
environment suitable for the host selected, prepare cell extract,
then add appropriate cell protease inhibitors. The desired protein
may be purified using standard procedures such as chromatography,
electrophoresis or density gradient centrifugation.
[0134] The above methods are also applicable to purifying proteins
from their native source.
[0135] Procedure for Purification of Proteins that Form Inclusion
bodies. U.S. Pat. No. 5,989,554 provides a general method for
isolating and purifying proteins that form inclusion bodies in
cells. First, the cells are lysed by enzymatic or mechanical means
in a buffer. The preferred method is sonication, although any other
lysis method will work, as long as lysis is complete and DNA and
RNA are sufficiently fragmented so as not to pellet upon
centrifugation. Preferred buffers contain Tris buffer at pH 7-8,
isotonic saline, and dithiothreitol (DTT) to maintain all cell
proteins in a reduced state. After sonication, detergent is added
to the mixture to solubilize most lipids and proteins, and the
mixture is centrifuged; it is preferred to use a centrifuge speed
of greater than 10,000 g for 10 minutes. The desired protein is
then found in the pellet fraction at a high degree of purity.
[0136] Higher purity is usually obtained by washing the pellet in a
second wash solution, often containing a different agent or
detergent. Washing is accomplished by resuspending the pellet in
the fresh buffer followed by centrifugation as above. The preferred
first detergent is sodium deoxycholate (NaDOC), and the second
preferred detergent is Triton X-100.
[0137] After the detergent washes, the pellet can be washed either
with the above buffer or with phosphate-buffered saline (PBS) to
remove trace detergent, then resuspended in a volume of a desired
buffer for storage or use in any of the ways described elsewhere,
including immunization.
[0138] The proteins purified by the above method are ready for use
in any or all of the applications contemplated in the invention,
including but not limited to the following; immunization of
animals, use as an adjuvant, coupling to other ligands, use as a
protease inhibitor, immobilization on hydrophobic surfaces, use as
an enzyme substrate, and use in peptide production after
cleavage.
[0139] As discussed earlier, this method is labor-intensive and
does not provide large quantities of active protein.
[0140] Novel Method for Purifying Proteins that Form Inclusion
Bodies. The present invention is based on the development of a
single-column purfication method for preparing milligram quantities
of >95% pure, active 27 kDa NIa proteinase. The method comprises
the following steps; [0141] a) obtaining cells expressing the
polypeptide; [0142] b) lysing the cells; [0143] c) pelleting
inclusion bodies by centrifugation of the lysed cells; [0144] d)
centrifuging the inclusion bodies; [0145] e) solubilizing the
inclusion bodies in buffer containing solubilization agent; [0146]
f) centrifuging the solubilized inclusion bodies to obtain a
supernatant containing the polypeptide; [0147] g) loading the
supernatant over a single denaturing column; [0148] h) collecting
the proteinase; and [0149] i) renaturing the collected polypeptide.
Preferably, the cells are lysed by incubating for 30 min at
4.degree. C. in 50 mM Tris-Cl, pH 8.0, 300 mM NaCl, 500 .mu.g/ml
lysozyme, 200 .mu.g/ml DNase I, and protease inhibitors (such as
PMSF and leupeptin); followed by three freeze-thaw cycles; addition
of Triton X-100 to a final concentration of 1%; and vortexing.
Preferably, following lysis the inclusion bodies are pelleted by
centrifugation at 10,000.times.g. Preferably, the inclusion bodies
are then solubilized with 6 M GuHCl, 100 mM NaH.sub.2PO.sub.4 and
10 mM Tris-Cl, pH 8.0. Preferably, before loading on the denaturing
column the solubilized inclusion bodies are centrifuged for 20
minutes at 15,000.times.g. Preferably, the denaturing column
contains Ni-NTA (nicke-nitilotiacetic acid-agarose) Superflow resin
(Qiagen). Preferably, the proteinase is eluted with 6 M urea, 100
mM NaH.sub.2PO.sub.4, and 10 mM Tris-Cl, pH 4.5, and the collected
proteinase fraction is adjusted with 10 N NaOH to pH 8.5, and
renatured during dialysis for 4-8 hours in storage buffer (100 mM
Tris-Cl, pH 8.5, 500 mM NaCl, 10% Glycerol, 5 mM DTT, 0.5 mM EDTA).
Preferably, as a final step, the dialyzed proteinase fraction is
centrifuged for 30 minutes at 15,000.times.g at 4.degree. C. to
collect the supernatant containing the active renatured
proteinase.
[0150] Other proteinase inhibitors, methods for lysing the cells,
buffers, solubilizing agents, and method of renaturing the purified
protein are well known to the skilled artisan, and some are
described in the previous sections of the specification. It is also
pointed out that any host cell that expresses large quantities of
the protein of interest in the form of inclusion bodies or any cell
that endogenously expresses the protein in the form of inclusion
bodies are useful for purifying large quantities of the protein of
interest.
[0151] In a preferred embodiment, the protein to be isolated in
large quantities is expressed in E. coli as a fusion protein
comprising in addition to the protein, a proteinase cleavage site,
and a fusion partner. Proteinase cleavage sites, as well as fusion
partner or carrier protein, are well known to the skilled artisan
and have been described in detail in other sections of the present
specification. A preferred proteinase cleavage site is the site
recognized by the 27 kDa NIa proteinase and the preferred carrier
protein is six His tags. The histidines enables binding to the
Ni-NTA column. The 27 kDa NIa proteinase, unlike other proteinases,
exhibits high specificity insensitivity to many proteinase
inhibitors used in protein purification, and efficient cleavage
under broad range of temperatures (Polayes et al., 1994). Moreover
the protein to be purified is easily separated from the carrier
peptide and the 27 kDa NIa proteinase.
[0152] In light of the foregoing general discussion, the specific
examples presented below are illustrative only and are not intended
to limit the scope of the invention. Other generic and specific
configurations will be apparent to those persons skilled in the
art.
EXAMPLES
Example 1
Cloning
[0153] A wild type 27 kDa NIa nucleic acid was generated by PCR
using pTL-5495 (ATCC 45036) as the template, 5TEVPI: 5'-CAT CAG CGG
GCC ATG GCT GAA AGC TTG TTT AAG-3'(SEQ ID NO: 2) as the 5' primer,
and 3TEVHI; 5'-CTG ATG CAC GGA TCC TCA TTA ATG GTG ATG GTG ATG GTG
CAA TTG CGA GTA GAC TAA TTC ACT CAT G-3' (SEQ ID NO: 3) as the 3'
primer. This nucleic acid translates to a proteinase with a
C-terminal His.sub.6 tag. The mutant 27 kDa NIa nucleic acid was
generated by sequential PCR reactions using pTL-5495 as the
template for the first reaction with 5TEVPI as the 5' primer and
TEVPSN: 5'-GAG TTG AGT TGC TTC TTT GAC TGG CTG AAA GGG TTC TTC AGG
TTT GTT CAT GAA AAC TTT GTG GCC-3' (SEQ ID NO: 4) as the 3' primer
to introduce the S219N mutation at the internal self-cleavage site.
The resultant PCR product was used as the template in a second PCR
reaction using the 5' primer 5TEVPI and the 3' primer 3TEVHI. The
nucleic acids were ligated into the pET15b expression vector
(Novagen) using the NcoI and BamHI sites and transformed into E.
coli DH5a (GibcoBRL) competent cells. Plasmids, pTPWT (wild type 27
kDa NIa proteinase) and pTPSN (mutant form with Ser to Asn
mutation), were sequenced for accuracy and transformed into BL21
(DE3) (Novagen) competent cells for expression.
Example 2
Proteinase Expression and Purification
[0154] The wild-type and mutant form 27 kDa NIa proteinases were
expressed in BL21 (DE3) cells grown at 37.degree. C. in Luria Broth
with 100 .mu.M Ampicillin to optical density 600 >0.7. Cultures
were induced with 400 .mu.M IPTG for >4 hours. Cell pellets were
harvested by centrifugation, resuspended in 50 ml buffer containing
50 mM Tris-Cl, pH 8.0 and 300 mM NaCl per liter of cell culture,
and stored at -80.degree. C. Lysis and purification of the soluble
fraction containing the 27 kDa NIa proteinase were performed as
described by Parks et al. (1995) through the Ni-NTA agarose
purification step, except 10% glycerol, 300 mM NaCl, and 5 mM
.beta.ME were included in all buffers, and the Ni-NTA agarose
column was washed with buffer containing 10 mM imidizole and eluted
with buffer containing 400 mM imidizole.
[0155] For inclusion body purfiication, cell suspensions were
thawed in cool water and the buffer was adjusted to give a final
concentration of 500 .mu.g/ml lysozyme (Sigma), 200 .mu.g/ml DNASE
I (Boehringer Mannheim), 50 .mu.g/ml PMSF, 10 .mu.g/ml Leupeptin
(Boehringer Mannheim), 20 mM MgSO.sub.4, and 2 mM CaCL.sub.2. The
cells were lysed as follows: rocking for 30 minutes at 4.degree.
C., followed by 3 freeze-thaw cycles, lysed the cells; adding
Triton x-100 to a final concentration of 1%; and vortexing. The
inclusion bodies were pelleted by centrifugation at 10,000.times.g.
Purified inclusion bodies were solubilized in a buffer containing 6
M GuHCl, 100 mM NaH.sub.3PO.sub.4 and 10 mM Tris-Cl, pH 8.0, and
stored at -80.degree. C.
[0156] Denaturing column chromatography was done at 4.degree. C.
using 10 ml Ni-NTA Superflow resin (Qiagen) per liter cell culture.
The column was equilibrated with 10 column volumes (cv) of
equilibration buffer (6M Urea, 100 mM NaH.sub.2PO.sub.4, and 10 mM
Tris-Cl, pH 8.0). The solubilized inclusion bodies were thawed in a
65.degree. C. bath and centrifuged 20 minutes at 15,000.times.g at
4.degree. C. The supernatant was loaded onto the column by gravity
flow. The column was washed with 4 cv equilibration buffer, then 6
cv wash buffer (6 M Urea, 100 mM NaH.sub.2PO.sub.4, and 10 mM
Tris-Cl, pH 6.3). The proteinase was eluted in elution buffer (6 M
Urea, 100 mM NaH.sub.2PO.sub.4, and 10 mM Tris-Cl, pH 4.5) with a 5
minute column incubation between each fraction until a total of 6
fractions were collected. Fractions containing the 27 kDa NIa
proteinase were pooled, adjusted to pH 8.5 with 10 N NaOH, dialyzed
4-8 hours in storage buffer (100 mM Tris-Cl, pH 8.5, 500 mM NaCl,
10% Glycerol, 5 mM DTT, and 0.5 mM EDTA), and centrifuged 30
minutes at 15,000.times.g at 4.degree. C. The supernatant,
containing active, renatured 27 kDa NIa proteinase was separated
from the pellet, containing precipitated 27 kDa NIa proteinase, and
both were stored at -80.degree. C. The pellet is successively
resuspended in Equilibration Buffer and redialyzed to obtain more
renatured, active proteinase was needed.
[0157] The 27 kDa NIa proteinase preparations were quantitated
using the Bradford Assay (Biorad) using BSA as a standard. The
yield of active 27 kDa NIa proteinase from the soluble preparation
was estimated to be less than 10% of the total protein as this
sample was not assayed for activity. For the insoluble preparation,
the active 27 kDa NIa yield is reported as milligrams of proteinase
obtained from the first renaturation of the eluate from the Ni-NTA
agarose column.
Example 3
Activity Assays
[0158] A 17 kDa substrate containing the target cleavage site
Gln-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 5) produces a .about.15 kDa
peptide when cleaved with commercially available 27 kDa NIa
proteinase (GibcoBRL). Substrate cleavage act5ivity was assayed in
30 .mu.l reactions with 100, 250, or 500 .mu.M substrate and 0.75
.mu.g proteinase in assay buffer (50 mM Tris-Cl, pH 8.0, 1 mM DTT,
and 0.5 mM EDTA) incubated at 30.degree. C. for 1 hour. Samples
were electrophoresed in 15% SDS-PAGE gels, stained with Coomassie
Blue, and protein bands were quantitated using a gel documentation
system (Kodak). The net intensities of full-length and cleaved
substrate bands were used to calculate the specific activity for
the substrate at each substrate concentration.
[0159] Self-cleavage activity was assayed in 40 .mu.l reactions
containing 7.5 .mu.g 27 kDa NIa proteinase in 50 mM Tris-Cl, pH
8.0, 1 mM DTT, and 0.5 mM EDTA, incubated at 4.degree. C.,
-20.degree. C. and -80.degree. C. At 0.5, 1, 2, and 5.5 weeks, 10
.mu.l sample was removed and boiled with denaturing SDS-PAGE
loading buffer, loaded onto 15% SDS-PAGE gels, and quantitated as
per above. The net intensities of full-length and cleaved
proteinase bands were used to calculate the percent proteinase
cleaved at each time point.
Results
[0160] pTPWT and pTPSN encode peptides with respective molecular
weights of 28,563 Daltons (about 28.5 kDa) and 28,590 Daltons
(about 28.5 kDa). During cloning, the proteinase N-terminus was
altered from wild type Gly-Glu-Ser- to Met-Ala-Glu-Ser- (SEQ ID No;
6) and a -Leu-His-His-His-His-His-His (SEQ ID NO: 7) tag was added
to the C-terminus.
[0161] Soluble preparations of 27 kDa NIa proteinase yielded
minimal quantities of proteinase (Table 1). 27 kDa NIa proteinase
detected as a band in cell lysate is missing in clarified lysate
but present in the inclusion body preparation (FIG. 1, lanes 1-3
and 7-9), Ni-NTA agarose purification under denaturing conditions
yields purer proteinase than purification under soluble conditions
(FIG. 1, lanes 4, 5, 10, and 11). In samples containing 4.5 .mu.g
total protein, a single contaminant is detected in renatured 27 kDa
NIa proteinase after the denaturing purification indicating >95%
purity (FIG. 1, lanes 13-15). Final yields of >10 mg renatured,
active 27 kDa NIa proteinase were regularly obtained after a single
renaturation step for both TPWT and TPSN (Table 1). Final
concentrations of several renatured preps indicate a maximum
solubility of .about.1.5 mg/ml (data not shown).
TABLE-US-00001 TABLE 1 Purification Yields for Soluble vs Insoluble
Procedures (per liter cell culture). Total Total Ni- protein Ni-
NTA-pure active NTA load protein rTEVP % (mg) (mg) (mg)* yield **
TPWT Soluble prep 977 4.08 <0.41 0.04 Insoluble prep 80 30.29
>10.96 1.04 TPSN Soluble prep 946 3.67 <0.37 0.04 Insoluble
prep 81 32.35 >12.84 1.25 *Active rTEVP for the soluble preps
are estimated to be <10% of total purified protein. Data
reported for insoluble preps is reported as greater than the
quantity of protein obtained from a single round of renaturation.
** % yield active rTEVP from total cellular protein Note: Yields of
total protein, active 27 kDa NIa proteinase, and overall percent
yield are shown for both the soluble and insoluble purification
protocols. Protein quantitation at each purification step was
estimated using the Bradford assay with a BSA standard curve.
[0162] The specific activity for substrate cleavage is defined as
the picomole substrate cleaved per picomole 27 kDa NIa proteinase
in 1 hour at 30.degree. C. The data indicate that TPWT and TPSN
have similar activity to each other and to the commercially
available proteinase at all substrate concentrations tested (FIG.
2).
[0163] Both the commercially available 27 kDa NIa proteinase and
purified TPWT contain the truncated self-cleavage product in the
final preparation, while the TPSN preparation contains only
full-length proteinase (FIG. 1, lanes 13-15). The self-cleavage
activity of TPWT is similar to the commercially available
proteinase at 4.degree. C. and -20.degree. C. TPSN has reduced
self-cleeavage activity at 4.degree. C., and no self-cleavage
activity at -20.degree. C. (FIGS. 3, 4a, and 4b). None of the
proteinase samples teseted exhibited significant self-cleavage
activity at -80.degree. C. during our 5.5 week assay (data not
shown).
CONCLUSION
[0164] The present invention shows that wild type, as well as
mutant, histidine-tagged 27 kDa NIa constructs can be induced to
express >95% of the proteinase in the insoluble fraction (FIG.
1). The present invention provides a novel method of denaturing
purification of the insoluble fraction followed by renaturation of
the peptide that yields up to 10 times as much active 27 kDa NIa
proteinase as the soluble preparation reported by Parks et al
(1995). This allows for stock preparations of >95% pure, active
27 kDa NIa proteinase to be made for general use.
[0165] Parks et al. (1995) has shown that the truncated form of 27
kDa NIa proteinase has significantly less substrate cleavage
activity than the full-length form. The proteinase continues to
cleave itself over time when stored at 4.degree. C. and -20.degree.
C., potentially reducing the quantity of fully active enzyme in an
27 kDa NIa proteinase stock over time. Self-cleavage appears to be
arrested at -80.degree. C.
[0166] The Ser219.fwdarw.Asn mutation in the mutant form of the 27
kDa NIa proteinase, provided by the present invention,
significantly inhibits self-cleavage activity, allowing for
increased yields of full-length, fully active 27 kDa NIa
proteinase, from either soluble or insoluble preps. The present
invention also permits long-term storage of 27 kDa NIa stocks at
-20.degree. C., and short term storage at 4.degree. c.
[0167] The 27 kDa proteinase and its mutant form are valuable tools
for protein purification protocols because of its target site
spcificity and its activity under a wide variety of conditions. The
present invention by utilizing a denaturing preparation of the TPSN
mutant, enables production of large stocks of rTEVP with consistent
activity characteristics.
[0168] It should be understood that the foregoing discussion and
examples merely present a detailed description of certain preferred
embodiments. It therefore should be apparent to those of ordinary
skill in the art that various modifications and equivalents can be
made without departing from the spirit and scope of the invention.
All journal articles, other references, patents, and patent
applications that are identified in this patent application are
incorporated by reference in their entirety.
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[0178] Dougherty, W. G. and Hiebert, E. (1980) Translation of
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T. D. (1988) Biochemical and Mutational Analysis of a Plant Virus
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Sequence CWU 1
1
717PRTPotyvirusmisc(2)..(2)any amino acid 1Glu Xaa Xaa Tyr Xaa Gln
Xaa 1 5 233DNAPotyvirus 2catcagcggg ccatggctga aagcttgttt aag
33367DNAPotyvirus 3ctgatgcacg gatcctcatt aatggtgatg gtgatggtgc
aattgcgagt agactaattc 60actcatg 67466DNAPotyvirus 4gagttgagtt
gcttctttga ctggctgaaa gggttcttca ggtttgttca tgaaaacttt 60gtggcc
6657PRTPotyvirus 5Glu Asn Leu Tyr Phe Gln Gly 1 5 64PRTPotyvirus
6Met Ala Glu Ser 1 77PRTPotyvirus 7Leu His His His His His His 1
5
* * * * *