U.S. patent application number 10/630932 was filed with the patent office on 2004-02-05 for method of purifying protein from inclusion bodies.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Gentz, Reiner L., Li, Yuling, Oelkuct, Mark.
Application Number | 20040024186 10/630932 |
Document ID | / |
Family ID | 28792534 |
Filed Date | 2004-02-05 |
United States Patent
Application |
20040024186 |
Kind Code |
A1 |
Li, Yuling ; et al. |
February 5, 2004 |
Method of purifying protein from inclusion bodies
Abstract
The present invention relates to processes for the purification
of proteins. More specifically, methods for solubilizing and
purifying proteins expressed in an insoluble form using low
concentrations of chaotropic agents, such as guanidine salts, are
provided. Also provided are methods for refolding proteins
solubilized according to the present invention.
Inventors: |
Li, Yuling; (Germantown,
MD) ; Oelkuct, Mark; (Frederick, MD) ; Gentz,
Reiner L.; (Belo Horizonte-Mg, BR) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
9410 KEY WEST AVENUE
ROCKVILLE
MD
20850
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
28792534 |
Appl. No.: |
10/630932 |
Filed: |
July 31, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10630932 |
Jul 31, 2003 |
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09215160 |
Dec 18, 1998 |
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6632425 |
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09215160 |
Dec 18, 1998 |
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08821637 |
Mar 20, 1997 |
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5912327 |
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Current U.S.
Class: |
530/351 |
Current CPC
Class: |
C07K 1/113 20130101;
C07K 14/50 20130101; C07K 1/36 20130101; C07K 14/521 20130101 |
Class at
Publication: |
530/351 |
International
Class: |
C07K 014/52 |
Claims
What is claimed is:
1. A method for recovering a target protein from inclusion bodies,
comprising: (a) treating the inclusion bodies with a chaotropic
agent at a concentration of about 0.7 to about 3.5M to solubilize
the target protein; and (b) recovering the target protein.
2. The method according to claim 1, where said chaotropic agent is
present at a concentration of about 1 to about 2 M.
3. The method according to claim 1, wherein said chaotropic agent
is selected from the group consisting of a guanidine salt and
urea.
4. The method according to claim 3, wherein said guanidine salt is
selected from the group consisting of guanidine hydrochloride and
guanidine isothiocyanate
5. The method according to claim 1, wherein the inclusion bodies
are obtained by lysing a cell selected from the group consisting of
a bacterial microorganism, insect cells, mammalian cells and yeast
cells.
6. The method according to claim 1, where the target protein is a
chemokine.
7. The method according to claim 1, wherein, the target protein is
refolded prior to recovering the target protein.
8. The method according to claim 1, wherein, in step (b), recovery
of the target protein includes subjecting the target protein to
liquid chromatographic purification followed by tandem
chromatography.
9. The method according to claim 1, wherein, in step (b), recovery
of the target protein includes subjecting the target protein to
microfiltration followed by ultrafiltration.
10. The method according to claim 9 wherein, recovery of the target
protein further includes refolding the target protein and
subjecting the refolded target protein to liquid chromatographic
purification.
11. The target protein provided by the method according to claim
1.
12. The target protein according to claim 7, selected from the
group consisting of MPIF-1, MPEF-1d23, MIP-1.alpha., M-CIF,
MIP-4,Ck-.beta.-13, Ck-.alpha.-4, and FGF-13.
13. The target protein according to claim 8, selected from the
group consisting of MPIF-1, MPIF-1d23, MIP-1.alpha., M-CIF, MIP-4,
Ck-.beta.-13, Ck-.alpha.-4, and FGF-13.
14. The target protein according to claim 9, selected from the
group consisting of MPIF-1, MPIF-1d23, MIP-1.alpha., M-CIF, MIP-4,
Ck-.beta.-13, Ck-.alpha.-4, and FGF-13.
15. The method according to claim 1, where the recovered target
protein is greater than 80% pure.
16. The method according to claim 8, where the recovered target
protein contains an endotoxin level of about 0.1 to about 1 EU/mg
of protein.
17. The method according to claim 8, wherein said recovered target
protein includes refolded target protein.
18. A method of recovering a secreted target protein comprising:
(a) subjecting the target protein to liquid chromatographic
purification; (b) subjecting the target protein of (a) to tandem
chromatography; and (c) recovering the target protein.
19. The method according to claim 18, where the target protein is a
chemokine.
20. The purified target protein according to claim 19, selected
from the group consisting of MPIF-1, MPIF-1d23, MIP-1.alpha.,
M-CIF, MIP-4, Ck-.beta.-13 Ck-.alpha.-4 and FGF-13.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/215,160, filed Dec. 18, 1998, which is a divisional of U.S.
application Ser. No. 08/821,637, filed Mar. 20, 1997 (now U.S. Pat.
No. 5,912,327).
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to processes for the
purification of proteins. More specifically, methods for
solubilizing and purifying proteins expressed in an insoluble form
using low concentrations of chaotropic agents, such as guanidine
salts, are provided. Also provided are methods for refolding
proteins solubilized according to the present invention.
[0004] 2. Related Art
[0005] The advent of recombinant DNA technology has brought about
an entirely new generation of protein products. The ability to
clone and express proteins in bacteria, yeast and mammalian cells
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 often 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 .mu.m in diameter and
largely composed of recombinant protein, that can be separated from
soluble bacterial proteins by low-speed centrifligation after cell
lysis (Schoner, et al. Biotechnology 3:151-154 (1985)).
[0006] 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.
[0007] 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).
[0008] Contrary to the teachings of the prior art, the inventors
have discovered that low concentrations of guanidine salts can be
used to solubilize biologically active (i.e., correctly folded)
proteins and extract this population of the protein from a
heterogeneous protein mixture localized in inclusion bodies. The
methods disclosed herein are particularly useful for the
purification of chemokines.
SUMMARY OF THE INVENTION
[0009] The present invention relates to the use of low
concentrations of guanidine salts to solubilize inclusion bodies
comprising target proteins. The purification method of the present
invention results in a highly homogeneous product with no
aggregated form of the target protein and can be easily scaled up
and adapted for cGMP manufacturing. Further, the recovered product
has a high purity, extremely low endotoxin levels as well as low
levels of residual DNA.
[0010] According to the invention, inclusion bodies are released
from cells by lysis, optionally washed to remove cellular
components prior to extraction, and extracted with solutions
containing low concentrations of guanidine salts.
[0011] More specifically, the invention provides for methods for
solubilizing inclusion bodies by treatment with guanidine salts at
concentrations of about 0.7 to about 3.5 M. The present invention
also provides methods for solubilizing inclusion bodies by
treatment with guanidine salts at concentrations of about 1 to
about 2 M.
[0012] In another aspect, methods are provided for refolding target
proteins which have been solubilized using guanidine salts. These
methods involve the rapid dilution of guanidine salt extracts and
optionally employ agents which facilitate target protein refolding.
The invention further relates to the purification of solubilized
target proteins using tandem column techniques.
[0013] The methods of the invention have the advantage of offering
uniformity of equipment requirements for any desired product. While
conditions required for optimum efficiency of solubilization and
refolding will vary with each protein, the present invention is
applicable, with only minor modification, for any protein which
forms inclusion bodies. The modifications required to achieve
optimum solubilization and refolding conditions for specific
proteins are disclosed herein or will be readily apparent to the
skilled artisan after reading the present specification.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1. SDS-PAGE analysis of in process samples of MPIF-1d23
under reducing conditions. The gel was stained with Coomassie blue.
Lane 1 shows the molecular weight standards. Lane 2: Total protein
from IPTG induced MPIF-1d23 transformed E. coli. Lane 3: Inclusion
body. Lane 4: 0.16 .mu.m filtered refold. Lane 5: HS purified
MPIF-1d23. Lane 6: HQ/CM purified MPIF-1d23.
[0015] FIG. 2. SDS-PAGE analysis of in process samples of MIP-4
(CK-beta-07) under reducing conditions. The gel was stained with
Coomassie blue. Lane 1 shows the molecular weight standards. Lane
2: Total protein from IPTG induced MIP-4 (CK-beta-07) transformed
E. coli. Lane 3: Inclusion body. Lane 4: 0.16 .mu.m filtered
refold. Lane 5: HS purified MIP-4 (CK-beta-07). Lane 6: HQ/CM
purified MIP-4 (CK-beta-07).
[0016] FIG. 3. SDS-PAGE analysis of 5 .mu.g purified MCIF-1 under
reducing conditions. The gel was stained with Coomassie blue. Lane
1 shows the molecular weight standards.
[0017] FIG. 4. SDS-PAGE analysis of 5 .mu.g purified MIP-4
(CK-beta-07) under reducing conditions. The gel was stained with
Coomassie blue. Lane 1 shows the molecular weight standards.
[0018] FIG. 5. SDS-PAGE analysis of 5 .mu.g purified MPIF-1 under
reducing conditions. The gel was stained with Coomassie blue. Lane
1 shows the molecular weight standards.
[0019] FIG. 6. SDS-PAGE analysis of 5 .mu.g purified MPIF-1d23
under reducing conditions. The gel was stained with Coomassie blue.
Lane 1 shows the molecular weight standards.
[0020] FIG. 7. SDS-PAGE analysis of 3 .mu.g purified MIP-1 alpha.
The gel was stained with Coomassie blue. Lane 1: Molecular weight
standards. Lane 2: 3 .mu.g MIP-1 alpha under reducing conditions.
Lane 3: blank. Lane 4: 3 .mu.g MIP-1 alpha under non-reducing
conditions.
[0021] FIG. 8. SDS-PAGE analysis of 5 .mu.g purified CK-alpha-04
under reducing conditions. The gel was stained with Coomassie blue.
Lane 1 shows the molecular weight standards.
[0022] FIG. 9. Silver stained SDS-PAGE analysis of purified
MPIF-1d23 under reducing conditions. The gel was loaded with the
indicated amounts of purified MPIF-1d23 and was silver stained.
Lane 1 shows the molecular weight standards.
[0023] FIG. 10: MPIF-1d23 inclusion body was prepared and
solubilized as described on page 23 steps (1)-(6) with the
indicated concentrations of GuHCl for 3 hours, separately. 20 .mu.l
of the GuHCl extracts were precipitated with Trichloride Acid (TCA)
and analyzed by SDS-PAGE under reducing conditions. The gel was
stained with coomassie blue.
[0024] FIG. 11: SDS-PAGE analysis of 5 .mu.g purified FGF-13. The
gel was stained with Coomassie blue. Lane 1: 5 .mu.g FGF-13 under
reducing conditions. Lane 2: Molecular weight standards. Lane 3: 5
.mu.g FGF-13 under non-reducing conditions.
[0025] FIG. 12. Purification of MPIF-1d23 through strong Cation
Exchange HPLC in a BioCad 250 HPLC workstation (PerSeptive
Biosystem). The refolded MPIF-1d23 sample was filtered through a
0.16 .mu.m tangential filtration unit, then applied to a POROS
HS-50 column. The HS column was washed with a buffer containing 50
mM sodium acetate, 300 mM sodium chloride, then eluted with 500 mM,
1000 mM, 1500 mM sodium chloride steps in 50 mM sodium acetate pH
6.0. The absorbance of 280 nm and conductivity were continuously
monitored. Active MPIF-1d23 was mainly present in the 0.5 M sodium
chloride fractions.
[0026] FIG. 13. Purification of MPIF-1d23 through Anion/Cation
Exchange tandem HPLC in a BioCad 60 HPLC workstation
(PerSeptiveBiosystem). The HS purifiedMPIF-1d23 sample was diluted
2-fold, then applied to a set of POROS HQ-50/CM-20 columns in a
tandem mode. Both columns were washed with a buffer containing 50
mM sodium acetate, 250 mM sodium chloride. The CM column was eluted
with a 10-20 column volume linear gradient of 250-1000 mM sodium
chloride in 50 mM sodium acetate pH 6.0. The absorbance of 280 nm,
260 nm and conductivity were continuously monitored. Active
MPIF-1d23 was present in the main peak.
[0027] FIGS. 14A and 14B show the effects of MPIF-1 concentration
on the growth and differentiation of HPP-CFC and LPP-CFC. A low
density population of mouse bone marrow cells was plated (1,500
cells/dish) in agar containing medium with or without the indicated
concentrations of the chemokines, but in the presence of IL-3 (5
ng/ml), SCF (100 ng/ml), IL-1 alpha (10 ng/ml), and M-CSF (5
ng/ml). Colonies were counted after 14 days. Data shown are pooled
from two independent experiments and are expressed as Mean +/-S.D.
The results show that MPIF-1 purified according to present
invention inhibits growth of LPP-CFC colonies in a dose-dependent
fashion.
[0028] FIG. 15 shows a comparison of MPIF-1 and MIP-1.alpha.
effects on colony formation by HPP-CFC and LPP-CFC. A low density
population of mouse bone marrow cells was plated (1,500 cells/dish)
in agar containing medium with or without the indicated chemokines
(100 ng/ml) plus standard cytokine cocktail as used for the HPP-CFC
and LPP-CFC assay. The number of colonies generated in the presence
of chemokines is expressed as a mean percentage of those produced
in the absence of any added chemokines. Data shown are pooled from
two independent experiments and are expressed as Mean +/-S.D. The
results demonstrate that MPIF-1 purified according to the present
invention inhibits colony formation by LPP-CFC, but not
HPP-CFC.
[0029] FIG. 16 shows the effect of MPIF-1 on the cytokine-induced
proliferation of human hematopoietic progenitor cells in vitro.
Hematopoietic progenitors were obtained by culturing human cord
blood CD34+ stem cells in the presence of IL-3 (10 ng/ml) and SCF
(50 ng/ml) for four days in liquid culture. These cells were then
plated (5,000 cells/well) in a 96-well plate in 0.2 ml of growth
medium containing the indicated concentrations of MPIF-1 either in
the presence or absence of a cytokine cocktail (CC2) consisting of
IL-3 & GM-CSF (1 ng/ml each), SCF (5 ng/ml) &
erythropoietin (3 U/ml). Cells were then allowed to grow in a
tissue culture incubator for six days at which point the numbers of
cells in each well was quantitated colorimetrically using the WST-1
reagent. Data from one out of three representative experiments are
shown as Mean absorbance +/-S.D. of assays that were performed in
triplicates. Note that 0.01 ng/ml represents no added MPIF-1.
[0030] FIG. 17 shows the effect of MPIF-1 on the 5-Fu-induced
cytotoxicity against HPP-CFC and LPP-CFC in in vitro liquid
cultures. Lin.sup.- populations of mouse bone marrow cells were
resuspended (1.times.10.sup.5 cells/ml) in a growth medium
containing IL-3 (5 ng/ml), SCF (50 ng/ml), GM-CSF (5 ng/ml), M-CSF
(5 ng/ml) and IL-1.alpha. (10 ng/ml) and 1 ml of this cell
suspension was dispensed into culture tubes. (1) A set of duplicate
cultures received no chemokine; (2) duplicate cultures with MPIF-1
at 100 ng/ml; and (3) duplicate cultures with an irrelevant protein
(i.e., MIP-4 (chemokine .beta.-7), a chemokine previously tested to
be not active in both HPP-CFC and LPP-CFC assay and is used as a
negative control) at 100 ng/ml. All cultures were incubated in a
tissue culture incubator for 48 hours, at which point one culture
from each set received 5-fluorouracil (5-Fu) at 100 .mu.g/ml and
incubation was continued for additional 24 hours. All cultures were
then harvested, washed three times with HBSS, and then assayed for
the presence of the HPP-CFC & LPP-CFC. Percent protection is
expressed as number of colonies detected in cultures incubated in
the presence of 5-Fu divided by the number of colonies found in
cultures incubated without 5-Fu.times.100. Data from one out of two
representative experiments are shown and are expressed as Mean
.+-.SD of assays that were performed in duplicates.
[0031] FIG. 18 shows the effect of MPIF-1 on the survival of human
hematopoietic progenitors from 5-Fu-induced cytotoxicity in vitro.
CD34+ cells (5.times.10.sup.4/ml) were cultured in 2 ml of growth
medium supplemented with IL-3 (10 ng/ml) and SCF (50 ng/ml) in the
absence of any chemokine (1) and in the presence of MIP-1.alpha.
(2) or MPIF-1 at 10 ng/ml (3). After allowing cells to grow for
four days, each culture was equally split into two cultures. One
culture of each set received 5-Fu at 25 .mu.g/ml and the other
served as a control. Both sets of cultures were then incubated for
one additional day. All cultures were then harvested, washed three
times with HBSS, and then assayed in triplicate in a 96-well cell
proliferation assay to determine proliferative potential in the
presence of IL-3 (10 ng/ml), GM-CSF (10 ng/ml), and SCF (50 ng/ml).
Plates were incubated in a tissue culture incubator for six days
and the total number of cells in each well was then determined
colorimetrically using WST-1 reagent. Percent protection equals
mean absorbance in the presence of 5-Fu/mean absorbance in the
absence of 5-Fu.times.100. Data from one out of two representative
experiments are shown as mean % protection .+-.S.D.
[0032] FIG. 19 shows calcium mobilization by MPIF-1 in THP-1 cells.
MPIF-1 induces calcium mobilization in human THP-1 cells in vitro.
THP-1 cells were exposed to 100 ng/ml MPIF-1 at the indicated time.
MPIF-1 induced a rapid mobilization of calcium in the THP-1
cells.
[0033] FIG. 20 shows calcium mobilization at various concentrations
of MPIF-1, MPIF-2, MIP-1.alpha. in THP-1 cells. THP-1 cells loaded
with Indo-1 were stimulated with various concentrations of MPIF-1,
MPIF-2 and MIP-1.alpha.. The maximal changes in intracellular
calcium concentration were measured.
[0034] FIG. 21 shows calcium mobilization at various concentrations
of MPIF-1, MPIF-2, MIP-1.alpha. in monocytes. Monocytes were
stimulated with various concentrations of MPIF-1, MPIF-2 and
MIP-1.alpha..
[0035] FIGS. 22A and 22B show the desensitization of THP-1 cells by
MPIF-1 and MIP-1.alpha.. The data shown in Panel A was obtained
using THP-1 cells stimulated with MPIF-1 followed by MIP-1.alpha..
The data obtained in Panel B was obtained using THP-1 cells
stimulated with MIP-1.alpha. followed by MPIF-1.
[0036] FIGS. 23A and 23B show the desensitization of monocytes by
MPIF-1 and MIP-1.alpha.. The data shown in Panel 23A was obtained
using Monocytes stimulated with MPIF-1 followed by MIP-1.alpha..
The data shown in Panel B was obtained using Monocytes stimulated
with MIP-1.alpha. followed by MPIF-1.
[0037] FIG. 24 shows the effect of MPIF-1 chemotaxis on PBMCs.
[0038] FIG. 25 shows the effect of MPIF-1 chemotaxis on
neutrophils.
[0039] FIG. 26 shows the effect of MPIF-1 chemotaxis on
monocytes.
[0040] FIG. 27 shows the effect of MPIF-1 chemotaxis on
T-lymphocytes.
[0041] FIG. 28 shows SDS-Page analysis of
Microfiltration-Ultrafiltration in process samples of MPIF-1d23
under reducing conditions. The gel was stained with coomassie blue.
Lane 1 shows the molecular weight standards. Lane 2 Total, protein
from IPTG induced MPIF-1d23 transformed E. coli post lysis. Lane 3
Microfiltration filtrate of a two times concentrate of total
protein from IPTG induced Id23 transformed E. coli post lysis. Lane
4 Microfiltration filtrate after two diafiltration wash volumes.
Lane 5 Microfiltration filtrate after five diafiltration wash
volumes. Lane 6 Remaining contaminants present in sample
supernatant after microfiltration wash. Lane 7 Inclusion body. Lane
8 Microfiltation starting sample supernatant of the 1.5 M Gu-HCl
solubilized sample. Lane 9 Microfiltration retentate after
filtration of the 1.5 M Gu-HCl solubilized sample. Lane 10
Ultrafiltration retentate of the 1.5 M Gu-HCl solubilized
sample.
[0042] FIG. 29 shows SDS-PAGE analysis of
Microfiltration-Ultrafiltration in process samples of MPIF-1d23
under reducing conditions. The gel was stained with coomassie blue.
Lane 1 shows the molecular weight standards. Lane 2 Ultrafiltration
filtrate after 2 diafiltration volumes with refold buffer. Lane 3
Ultrafiltration filtrate after 3 diafiltration volumes with refold
buffer. Lane 4 Ultrafiltration filtrate after 4 diafiltration
volumes with refold buffer. Lane 5 Ultrafiltration retentate pellet
post diafiltration refold. Lane 6 0.16 .mu.m filtered refold of the
Ultrafiltration diafiltration refold sample. Lane 7 HS flow
through. Lane 8 HS purified MPIF-1d23. Lane 9 HQ/CM purified
MPEF-1d23.
[0043] FIG. 30 shows the Microfiltration and Ultrafiltration
Process Equipment-Product Flow Diagram.
DETAILED DESCRIPTION OF THE INVENTION
[0044] The present invention provides solubilization and refolding
procedures which are applicable to recombinant proteins that form
inclusion bodies. The present invention provides methods which
employ low concentrations of guanidine salts (e.g., about 0.7 to
about 3.5 M) to solubilize active protein from inclusion bodies
which is contrary to the teachings of the prior art that
concentrations of 6-8 M guanidine salt are necessary for such
solubilization. As indicated above, the advantages of using the
methods of the present invention for the purification of target
proteins are that it is simple, low in cost, can be easily scaled
for use with large volumes and adapted for cGMP manufacturing.
Further, the present invention results in a highly homogeneous
biologically active product having high purity, low residual DNA
levels and extremely low endotoxin levels with no aggregated forms
of the target protein.
[0045] By "target protein" is intended secreted recombinant protein
or a recombinant protein localized in an inclusion body which is
the subject of a purification procedure.
[0046] In another aspect, by "target protein" is intended secreted
endogenous protein or endogenous protein localized in an inclusion
body wherein the endogenous protein's natural expression
characteristics have been modified using homologous recombination.
By "endogenous" protein is intended protein expressed from a gene
naturally occurring in the genome of a cell. Expressing protein
using homologous recombination, which is described in detail in
U.S. Pat. No. 5,272,071, WO 90/11354, WO91/06666, WO 91/06667, and
WO 95/31560 (each of which are herein incorporated by reference),
occurs without transfecting a cell with DNA that encodes the
protein of interest. Instead, the gene is identified within the
cell's genome and activated by inserting an appropriate regulatory
segment by homologous recombination such that the regulatory
segment is operatively linked to the gene and thereby modifies its
expression. Positive and/or negative selectable markers can also be
inserted to aid in selection of cells wherein the proper homologous
recombination events have occurred. As an additional embodiment,
such naturally occurring genes can be amplified for enhanced
protein expression, whether the gene is normally transcriptionally
silent and has been activated by the integrated regulatory segment,
or endogenously expresses product.
[0047] By "refolded" is intended that a conformational form has
been produced that exhibits at least one biological activity of the
protein. Preferably, protein purified according to the present is
refolded to its native conformation.
[0048] By "recombinant protein" is intended protein expressed in a
host cell from a recombinant nucleic acid molecule.
[0049] By "inclusion body" is intended an insoluble protein
aggregate produced by a microorganism (e.g., a bacterium) or other
host cell (e.g., an insect or mammalian host cell). Examples of
inclusion bodies are recombinantly expressed proteins.
[0050] By "biological activity" is intended an activity normally
associated with a protein. For example, chemokines (intercrine
cytokines) exhibit a wide variety of biological activities. A
hallmark feature is their ability to elicit chemotactic migration
of distinct cell types, including monocytes, neutrophils, T
lymphocytes, basophils and fibroblasts. Many chemokines also have
proinflammatory activity and are involved in multiple steps during
an inflammatory reaction. These activities include stimulation of
histamine release, lysosomal enzyme and leukotriene release,
increased adherence of target immune cells to endothelial cells,
enhanced binding of complement proteins, induced expression of
granulocyte adhesion molecules and complement receptors, and
respiratory burst. Certain chemokines have also been shown to
exhibit other activities such as suppressing hematopoietic stem
cell proliferation, inhibiting endothelial cell growth, and
proliferating keratinocytes. Of course, activities normally
associated with a vast number of other proteins are known in the
art.
[0051] In one aspect, the invention provides a method for
recovering a target protein from inclusion bodies which involves
(1) treating inclusion bodies from lysed host cells with about 0.7
to about 3.5 M chaotropic agent to solubilize the protein; and (2)
recovering the protein. The recovered, solubilized protein may then
be purified using conventional techniques. Optionally, if deemed
necessary, the target protein may be refolded prior to
purification.
[0052] Also provided by the present invention is a method for
solubilizing protein localized in inclusion bodies using
concentrations of guanidine salt of about 1 to about 2 M. As one
skilled in the art would recognize, each individual protein will be
solubilized at a particular guanidine salt concentration.
[0053] In another aspect of the present invention, a series of
solubilization steps is provided to separate target proteins from
host cell contaminants. Initially, the cells containing the target
protein are lysed and the homogenate is centrifuged to pellet the
inclusion bodies. Preferably, the pellet is then washed (e.g., with
a buffer containing 50 mM Tris, 25 mM EDTA and 0.5 M NaCl) to
separate the inclusion bodies from cellular components. The
partially purified inclusion bodies are then solubilized with a
solution containing a guanidine salt (e.g., 1.5 M for 2-4 hours).
Solubilized target protein remains in the soluble phase after
centrifugation (e.g., 7,000.times.g). The supernatant is placed at
2-10.degree. C. overnight prior to the second centrifugation at
30,000.times.g. Optionally, the resulting pellet can be further
extracted overnight followed by re-centrifugation (e.g., at
30,000.times.g). The 30,000.times.g supernatant is referred to as
the guanidine salt extract which generally contains most of the
target protein originally present in the inclusion bodies. The
portion of the protein that remains in the pellet after such
extraction is generally difficult to refold and not biologically
active.
[0054] Non-limiting examples of the guanidine salts that can be
used in the process of the invention include guanidine
hydrochloride and guanidine isothiocyanate. Urea can also be used
as the chaotropic agent instead of the guanidine salts at
concentrations of about 1 to about 4 M. In addition, detergents may
be used to solubilize the target protein.
[0055] The inventors have shown that the present invention provides
solubilized, biologically active target protein of higher yield and
better purity than the processes known in the prior art. When the
recombinant proteins are produced in the bacterial cell, inclusion
bodies are formed that are heterogeneous in protein components.
This heterogeneity comprises many conformational forms of the
target protein that range from those which are correctly folded,
those which are partially correctly folded, and those which are
completely incorrectly folded. It is likely that the forms of the
target protein present in the inclusion bodies that are closest to
the correct protein conformation will be solubilized more readily
in lower concentrations of guanidine salts than will those forms
that are less correctly folded. Surprisingly, the present inventors
have obtained substantially higher yields of active protein using
the process of the present invention than that obtainable by
methods taught by the art that require higher concentrations (6-8
M) of guanidine salts for the solubilization of the inclusion body
protein. The methods taught by the art involving the use of high
concentrations of chaotropic agents may yield more protein but, in
contrast to the process of the present invention, most of the
protein is in an inactive form.
[0056] Although target proteins purified using the methods of the
present invention will generally be correctly folded, a certain
population of the isolated target protein may not be in a
completely proper conformation. Thus, the present invention also
provides methods for refolding proteins solubilized in the presence
of guanidine salts. These methods involve the rapid dilution of
guanidine salt extracts containing the solubilized target protein.
In one aspect of the present invention, the guanidine salt
extracted target protein is refolded by rapidly mixing the
guanidine salt extract with a large volume of a buffer containing
little or no guanidine salt (e.g., 20 volumes of 50-100 mM sodium
acetate pH 4.5, 150 mM sodium chloride, 2 mM EDTA).
[0057] After solubilization of inclusion bodies, the solubilized
proteins may be present in reduced form and may not contain
disulfide bonds found in the native protein. Several known methods
for regenerating native sulfide bonds include air oxidation
systems, glutathione renaturation systems, and renaturation using
mixed sulfides. These methods are described in Fischer, B. Biotech.
Adv. 12:89-101 (1994), which is herein incorporated by
reference.
[0058] Depending on the factors which contribute to the native
conformation of the target protein, it may be desirable to refold
the target protein either using a renaturing system or in the
presence of a reagent which assists in the formation of the target
protein's native conformation. Such reagents include reducing
agents, oxidizing agents and salts. Examples of such agents include
DTT, .beta.-mercaptoethanol, glutathione, cysteamine and cysteine.
Other such agents would be apparent to one skilled in the art.
Concentrations and incubation conditions for the use of these
agents with the present invention would also be apparent to one
skilled in the art. Generally, however, when DTT is used a protein
concentration of 100 .mu.g/ml to 500 .mu.g/ml of protein would be
treated with 5-100 mM DTT for about 24 hours at 2-25.degree. C.
Similarly, when .beta.-mercaptoethanol is used a concentration of
100 .mu.g/ml to 300 .mu.g/ml of protein may generally be treated
with 10 mM-200 mM .beta.-mercaptoethanol for about 24 hours at
2-25.degree. C. Similarly, when glutathione is used a concentration
of 100 .mu.g/ml to 300 .mu.g/ml of protein may generally be treated
with 1-10 mM oxidized and 9-90 mM reduced glutathione for about 24
hours at 2-25.degree. C. Thus, another aspect of the present
invention provides methods for refolding target proteins in the
presence of agents which aid in the formation of the protein's
native conformation.
[0059] The methods of the present invention can be applied to
solubilize and refold any target protein deposited in inclusion
bodies (i.e., cytokines, growth factors, enzymes, transcription
factors, etc.). Non-limiting examples of such proteins include
fibroblast growth factor 13 (FGF13), myeloid progenitor inhibitory
factor-1 (MPIF-1), myeloid progenitor inhibitory factor-1 having
the N-terminal 23 amino acids deleted (MPIF-1d23), macrophage
inhibitory protein-1.alpha. (MIP-1.alpha.), monocyte-colony
inhibitory factor (M-CIF), macrophage inhibitory protein-4 (MIP-4),
chemokine-.alpha.4 (ck-.alpha.4), chemokine .beta.-13 (ck
.beta.-13).
[0060] After solubilization and, optionally, refolding of target
proteins, these proteins can be recovered and purified by methods
well known in the art including ammonium sulfate or ethanol
precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, reverse phase chromatography, affinity
chromatography, hydroxylapatite chromatography and lectin
chromatography. Most preferably, high performance liquid
chromatography ("HPLC") is employed for purification.
[0061] Target proteins may be expressed in modified form, such as a
fusion protein, and may include heterologous functional regions.
For instance, a region of additional amino acids, particularly
charged amino acids, may be added to the N-terminus or C-terminus
of the target protein to improve stability and persistence in the
host cell, during purification, or during subsequent handling and
storage. Also, peptide moieties may be added to the target protein
to facilitate purification. Such regions may be removed prior to
final preparation of the target protein.
[0062] The present invention can also be used to purify His-tagged
proteins produced in an insoluble form. The bacterial expression
vector pQE9 (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,
91311) is one example of a vector which may be used to produce a
target protein containing a His tag. A DNA sequence encoding a
target protein can be inserted into this vector such that the
inserted sequence expresses the target protein with the His
residues covalently linked to the amino terminus of the protein.
These histidine residues allow for the affinity purification of the
target protein using a nickel-nitrilo-tri-acetic acid ("Ni-NTA")
affinity resin.
[0063] Vectors and Host Cells for Expression of Target Protein
[0064] Recombinant constructs for the expression of target protein
may be introduced into host cells using well known techniques such
as infection, transduction, transfection, transvection,
electroporation and transformation. The vector may be, for example,
a phage, plasmid, viral or retroviral vector. Retroviral vectors
may be replication competent or replication defective. In the
latter case, viral propagation generally will occur only in
complementing host cells.
[0065] The polynucleotides encoding the target protein may be
joined to a vector containing a selectable marker for propagation
in a host. Generally, a plasmid vector is introduced in a
precipitate, such as a calcium phosphate precipitate, or in a
complex with a charged lipid. If the vector is a virus, it may be
packaged in vitro using an appropriate packaging cell line and then
transduced into host cells.
[0066] Preferred are vectors comprising cis-acting control regions
to the polynucleotide of interest. Appropriate trans-acting factors
may be supplied by the host, supplied by a complementing vector or
supplied by the vector itself upon introduction into the host.
[0067] Vectors that provide for specific expression, include those
that may be inducible and/or cell type-specific. Particularly
preferred among such vectors are those inducible by environmental
factors that are easy to manipulate, such as temperature and
nutrient additives.
[0068] Expression vectors useful for the expression of target
proteins include chromosomal-, episomal- and virus-derived vectors,
e.g., vectors derived from bacterial plasmids, bacteriophage, yeast
episomes, yeast chromosomal elements, viruses such as
baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl
pox viruses, pseudorabies viruses and retroviruses, and vectors
derived from combinations thereof, such as cosmids and
phagemids.
[0069] The DNA insert containing the gene for the target protein
should be operatively linked to an appropriate promoter, such as
the phage lambda PL promoter, the E. coli lac, trp and tac
promoters, the SV40 early and late promoters and promoters of
retroviral LTRs, to name a few. Other suitable promoters will be
known to the skilled artisan. The expression constructs will
further contain sites for transcription initiation, termination
and, in the transcribed region, a ribosome binding site for
translation. The coding portion of the target transcripts expressed
by the constructs will preferably include a translation initiating
at the beginning and a termination codon (UAA, UGA or UAG)
appropriately positioned at the end of the polypeptide to be
translated.
[0070] As indicated, the expression vectors will preferably include
at least one selectable marker. Such markers include dihydrofolate
reductase or neomycin resistance for eukaryotic cell culture and
tetracycline, kanamycin or ampicillin resistance genes for
culturing in E. coli and other bacteria. Representative examples of
appropriate hosts include, but are not limited to, bacterial cells,
such as E. coli, Streptomyces and Salmonella typhimurium cells;
fungal cells, such as yeast cells; insect cells such as Drosophila
S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and
Bowes melanoma cells; and plant cells. Appropriate culture mediums
and conditions for the above-described host cells are known in the
art.
[0071] Among vectors preferred for use in bacteria expression of
target proteins include pQE70, pQE60, pQE6, pQE7 and pQE-9,
available from Qiagen; pBS vectors, Phagescript vectors, Bluescript
vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene;
and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from
Pharmacia or the pHE vector series developed at HGS, Inc. Among
preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and
pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia. Other suitable vectors will be readily
apparent to the skilled artisan.
[0072] Among known bacterial promoters suitable for expression of
target protein include the E. coli lacI and lacZ promoters, the T3
and T7 promoters, the gpt promoter, the lambda PR and PL promoters
and the trp promoter. Suitable eukaryotic promoters include the CMV
immediate early promoter, the HSV thymidine kinase promoter, the
early and late SV40 promoters, the promoters of retroviral LTRs,
such as those of the Rous sarcoma virus (RSV), and metallothionein
promoters, such as the mouse metallothionein-I promoter.
[0073] Introduction of the expression construct into the host cell
can be effected by calcium phosphate transfection, DEAE-dextran
mediated transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other methods. Such
methods are described in many standard laboratory manuals, such as
Davis et al., Basic Methods In Molecular Biology (1986).
[0074] Transcription of the DNA encoding the target protein by
higher eukaryotes may be increased by inserting an enhancer
sequence into the vector. Enhancers are cis-acting elements of DNA,
usually about from 10 to 300 bp that act to increase
transcriptional activity of a promoter in a given host cell-type.
Examples of enhancers include the SV40 enhancer, which is located
on the late side of the replication origin at bp 100 to 270, the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0075] For secretion of the translated target protein into the
lumen of the endoplasmic reticulum, into the periplasmic space or
into the extracellular environment, appropriate secretion signals
may be incorporated into the expressed target protein. The signals
may be endogenous to the target protein or they may be heterologous
signals.
[0076] The target protein may be expressed in a modified form, such
as a fusion protein, and may include not only secretion signals,
but also additional heterologous functional regions. For instance,
a region of additional amino acids, particularly charged amino
acids, may be added to the N-terminus of the polypeptide to improve
stability and persistence in the host cell, during purification, or
during subsequent handling and storage. Also, peptide moieties may
be added to the polypeptide to facilitate purification. Such
regions may be removed prior to final preparation of the
polypeptide. The addition of peptide moieties to polypeptides to
engender secretion or excretion, to improve stability and to
facilitate purification, among others, are familiar and routine
techniques in the art. A preferred fusion protein comprises a
heterologous region from immunoglobulin that is useful to
solubilize proteins. For example, EP-A-O 464 533 (Canadian
counterpart 2045869) discloses fusion proteins comprising various
portions of constant region of immunoglobin molecules together with
another human protein or part thereof. In many cases, the Fc part
in a fusion protein is thoroughly advantageous for use in therapy
and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). On the other hand, for
some uses it would be desirable to be able to delete the Fc part
after the fusion protein has been expressed, detected and purified
in the advantageous manner described. This is the case when Fc
portion proves to be a hindrance to use in therapy and diagnosis,
for example when the fusion protein is to be used as antigen for
immunizations. In drug discovery, for example, human proteins, such
as, hIL-5 has been fused with Fc portions for the purpose of
high-throughput screening assays to identify antagonists of hIL-5.
See, D. Bennett et al., Journal of Molecular Recognition, Vol.
8:52-58 (1995) and K. Johanson et al., The Journal of Biological
Chemistry, Vol. 270, No. 16:9459-9471 (1995).
[0077] Process for Purifying Target Proteins
[0078] The bench scale method of the invention for protein
production utilizes a series of solubilization procedures to
separate the target proteins from the host contaminants by: cell
lysis, 0.5M NaCl washes, to produce inclusion body containing
partially purified target protein.
[0079] In the one embodiment of the invention, the partially
purified inclusion bodies are subjected to further solubilization
with 1.5 M of a guanidine salt extraction for 2-4 hours. Target
protein is soluble and will stay in the soluble phase after
7,000.times.g centrifugation. Such sample is placed at 4.degree. C.
for further extraction overnight, centrifuged at 30,000.times.g.
The supernatant, called a 1.5M guanidine salt extract, contains
greater than 50% of the target proteins.
[0080] The 1.5M guanidine salt extracted target protein is refolded
by quickly mixing the guanidine salt extract with 10-20 volumes of
a buffer containing 50-100 mM sodium acetate pH 4.5, 150 mM sodium
chloride, 2 mM EDTA with 30-60 minutes of vigorous stirring. The
mixture is set at 2-10.degree. C. without mixing for 1-48 hours
prior to the chromatographic purification steps described
below.
[0081] The refolded target protein samples are clarified through a
0.16 .mu.m tangential filtration unit. The filtered sample was then
captured through a strong cation exchange column. Suitable column
materials are well known to those skilled in the art. Examples of
such materials include POROS HS-50 column, Mono S, S Sepharose, SP
Sepharose, Resource/Source S, Toyopearl S, Toyopearl SP. The cation
column is washed with a buffer containing 40 mM sodium acetate pH
4.5 to pH 6.0, 250 mM sodium chloride, eluted with steps of 500 mM,
1000 mM, 1500 mM sodium chloride. Target protein is eluted at the
500-1000 mM salt steps.
[0082] The cation column purified samples are diluted 4-fold, then
applied onto a strong anion/weak cation tandem columns. Suitable
column materials are well known to those skilled in the art.
Examples of such columns include POROS HQ-50/POROS CM-20.
Alternative materials for the strong anion column for the tandem
column include Mono Q, Q Sepharose, Resource/Source Q, DEAE,
Toyopearl Q. Alternative materials for the weak cation column for
the tandem column include CM Sepharose, Toyopearl CM. The target
protein will bind either to the anion or to the cation column
depending upon the particular target protein. As will be understood
by those skilled in the art, the arrangement of the column
materials in the tandem column chromatography step can be reversed
depending on which type of column material will bind target
protein. In the tandem chromatography step, contaminants such as
DNA, endotoxins and other proteins with lower pIs will bind to the
strong anion column and the chemokines will pass through and bind
to the weak cation column. For example, in the purification of
proteins such as chemokines, the tandem columns are run with the
chemokine sample passing over the strong anion column followed by
the weak cation column.
[0083] The tandem columns are washed with 40 mM sodium acetate pH
4.75-6.0, 150-250 mM sodium chloride. The column having the bound
target protein is eluted with a linear gradient of 250-1000 mM NaCl
in 10-20 column volumes. The eluted fractions are analyzed through
SDS-PAGE and the target protein containing peak fractions are
combined.
[0084] Using the process described above, 50% purity can be
achieved after the initial recovery and isolation of the inclusion
bodies. The refolded target proteins are about 60-70% pure with a
greater than 50% refolding efficiency. Indeed, the refolding
efficiency for chemokines is about 60-80%. The subsequent
chromatographic steps yield target proteins that were typically of
greater than 95% purity. No major contaminant bands are visualized
by commassie blue staining when 5 .mu.g of such purified protein is
analyzed on SDS-PAGE.
[0085] As the skilled artisan will recognize, additional
purification steps may be performed on the target protein using
techniques that are well known to those skilled in the art. For
example, additional chromatography steps such as size exclusion,
ion exchange, hydrophobic interaction, affinity, reverse phase may
be employed.
[0086] Although target proteins purified using the process of the
present invention will generally be correctly folded, a certain
population of the isolated target protein may not be in a
completely proper conformation. Thus, it may be desirable, in these
cases, to subject the target protein through a refolding process to
restore that population to its native form by treating with reagent
such as DTT, .beta.-mercaptoethanol, glutathione, cysteamine or
cysteine are present in the appropriate medium. In such cases,
generally a concentration 100 .mu.g/ml to 500 .mu.g/ml of protein
is treated with 5 mM-100 mM DTT for about 10-48 hrs at 2-25.degree.
C., preferably a concentration of 100 .mu.g/ml to 300 .mu.g/ml of
protein is treated with 5 mM -100 mM DTT for about 48 hrs at
2-10.degree. C. In addition, a concentration 100 .mu.g/ml to 500
.mu.g/ml of protein is treated with 10 mM-200 mM .beta.ME for about
10-48 hrs at 2-25.degree. C. Alternatively, a concentration 100
.mu.g/ml to 500 .mu.g/ml of protein is treated with 10 mM-100 mM
glutathione for about 10-48 hrs at 2-25.degree. C. The target
protein may, while in solution, be subjected to standard techniques
directed to the purification of the protein such as gel filtration
or ion exchange chromatography.
[0087] Industrial Scale Process for Purifying Target Proteins
[0088] The following provides an industrial scale manufacturing
process for the present invention. It will be apparent to those
skilled in the art that adjustments can be made to the process by
routine optimization to accommodate the increased size of finished
product desired. Within the stated process, there are steps that
require centrifugation for the recovery of product. Although
centrifugation is used in industrial scale manufacturing, the
product can alternatively be recovered using more economical and
scaleable techniques such as Microfiltration and Ultrafiltration
(Meagher et al, Biotechnology and Bioengineering 43:969-977
(1994)). This system has been employed for industrial scale
recovery, clarification, concentration, and diafiltration for E.
coli, mammalian and all insect cell expressed target proteins.
(FIG. 30).
[0089] The principles of filtration were successfully applied
within the stated process for inclusion body recovery,
solubilization and refold with no effect on the homogeneity,
identity, purity and activity of the target protein. For industrial
scale manufacturing the following parameters of the process can be
modified by the skilled artisan using routine optimization. The
following process is applicable to all proteins using this
invention.
[0090] Microfiltration is the use of a 0.45 .mu.m tangential flow
filter that retains large particles at approximately 0.45 .mu.m or
greater in diameter. This filter serves two purposes to wash
inclusion bodies and to filter product through during Gu-HCl
solubilization.
[0091] Ultrafiltration is the use of a 10,000 molecular weight cut
off (MWCO) tangential flow filter that retains particles at
approximately 10,000 kilodalton molecular weight or greater. The
filter serves two purposes: to capture Gu-HCl solubilized target
protein and to diafilter protein through refold.
[0092] (1) Bacterial cell paste is harvested by Microfiltration
using a Pall-Filtron Nylon 0.45 .mu.m tangential flow filter (such
filters for industrial scale manufacturing can also be obtained
from Millipore or AG Technology) and concentrated two fold. The E.
coli cell particles are retained by the filter at this step.
[0093] (2) Suspension is then lysed (2.times. in Microfluidizer
(6,000-12000 psi) to release inclusion bodies.
[0094] (3) The lysed sample with inclusion bodies are washed by
first diafiltering the sample with 4 diafiltration volumes of 100
mM Tris (pH 7.4); 25 mM EDTA and 0.5 M NaCl, followed by two
diafiltration volumes of 100 mM Tris (pH 7.4); 25 mM EDTA. The
inclusion bodies are retained by the filter at this step.
[0095] (4) Solubilization of target protein from inclusion bodies
is achieved by adding solid Gu-HCl directly to the washed inclusion
body suspension to a final concentration of about 1.5 M.
[0096] (5) The resulting supernatant contains target protein
released from inclusion bodies and is recovered by recirculating
the sample through the same Microfiltration device as in step
1.
[0097] (6) The product at this point passes through the 0.45 .mu.m
Microfiltration filter and is commonly referred to as filtrate. The
product is then retained and concentrated using the 10,000 Kd MWCO
Ultrafiltration device (such filters can be obtained from
Pall-Filtron, Millipore or AG Technology Hollow fiber Filters). The
Ultrafiltration filtrate is then passed back to the Microfiltration
sample in order to maintain a constant Microfiltration volume. The
recirculation process is maintained until >95% product has
passed through the Microfiltration device and is captured by the
Ultrafiltration device.
[0098] (7) The Ultrafiltration sample, containing about 80% target
protein, is stored at 2-10.degree. C. for 12-24 hours.
[0099] (8) The target proteins, such as chemokines, are refolded by
removing the 1.5 M Gu-HCl using the same Ultrafiltration device as
in step 6. The target proteins, such as chemokines are retained by
the 10000 Kd MWCO filter and the Gu-HCl is removed by diafiltering
the sample with 50-100 mM Sodium Acetate (pH4.5) 125 mM NaCl; 2 mM
EDTA. Approximately 4-6 diafiltration volumes are sufficient to
refold the protein.
[0100] (9) Liquid chromatographic purification is begun by either a
0.45 .mu.m or 0.22 .mu.m dead end filtration. However, a 0.16 .mu.m
tangential flow filtration can be used to filter out any
precipitate present that would foul dead end filtration.
[0101] At this point in the process liquid chromatography
purification is performed as stated above for the bench scale
process of the invention. For industrial scale manufacturing column
chromatography purification would be appropriately scaled to the
size of the manufacturing batch desired. The results of the
industrial scale process are shown in FIGS. 28 and 29. The skilled
artisan familiar in the practices of large scale cGMP manufacturing
would know how to adjust the parameters required to adapt this
process for their particular use.
[0102] Chemokines
[0103] Chemokines are one class of proteins which are generally
produced as inclusion bodies when expressed at a high level in
bacteria. Chemokines, also referred to as intercrine cytokines, are
a subfamily of structurally and functionally related cytokines.
These molecules are 8-14 kd in size. In general, chemokines exhibit
20% to 75% homology at the amino acid level and are characterized
by four conserved cysteine residues that form two disulfide
bonds.
[0104] The intercrine cytokines exhibit a wide variety of functions
as discussed above. In light of the diverse biological activities,
it is not surprising that chemokines have been implicated in a
number of physiological and disease conditions, including
lymphocyte trafficking, wound healing, hematopoietic regulation and
immunological disorders such as allergy, asthma and arthritis.
[0105] For example, the chemokines MPIF-1, MPIF-1d23, MIP-1.alpha.,
M-CIF, MIP-4, Ck-.beta.-13, and Ck-.alpha.-4 purified according to
the method of the present invention sed for therapeutic purposes,
such as, to protect bone marrow stem cells from erapeutic agents
during chemotherapy, to remove leukemic cells, to stimulate an
immune response, to regulate hematopojesis and lymphocyte
trafficking, treatment of psoriasis, solid tumors, to enhance host
defenses against resistant and acute and chronic infection, and to
stimulate wound healing. In addition, disorders that could be
treated with chemokines MPIF-1, MPIF-1d23, MIP-1.alpha., M-CIF,
MIP-4, Ck-.beta.-13, and Ck-.alpha.-4 include tumors, cancers, and
any disregulation of immune cell function including, but not
limited to, autoimmunity, arthritis, leukemias, lymphomas,
immunosuppression, sepsis, wound healing, acute and chronic
infection, cell mediated immunity, humoral immunity, inflammatory
bowel disease, myelosuppression, and the like.
[0106] A preferred embodiment of the present invention is outlined
below and comprises the following: cell lysis and inclusion body
suspension are provided by steps (1)-(3); inclusion body
solubilization is provided by steps (4)-(6); chemokine refolding is
provided by steps (7)-(8); and chemokine purification is provided
by steps (9)-(14).
[0107] (1) Bacterial cell paste is resuspended in buffer (100 mM
TRIS (pH 7.4); 50 mM EDTA);
[0108] (2) Suspension is lysed (2.times. in Microfluidizer
(4,000-12,000 psi)) to release inclusion bodies;
[0109] (3) Lysed sample with inclusion bodies is mixed with a NaCl
solution to bring the final concentration of NaCl to 0.5 M and then
centrifuged (7,000.times.g) 15 min;
[0110] (4) The pellet (containing chemokine target protein
inclusion bodies) is washed again with the same buffer as in step
(3). The pellet (i.e., inclusion bodies) is solubilized with using
1.5 M GuHCl (2-4 hours);
[0111] (5) The GuHCl solubilized sample is centrifuged
(7,000.times.g) and the resulting supernatant contains chemokine
protein released from inclusion bodies by step (4). A second
extraction of the 7,000.times.g pellet can be optionally performed
with a higher concentration GuHCl if desired;
[0112] (6) Supernatant is stored at 4.degree. C. for two hours and
the supernatant is optionally recentrifuged at up to 30,000.times.g
(80% chemokine);
[0113] (7) Chemokine is refolded by mixing the GuHCl extracted
supernatant with 10-20 volumes of buffer (50-100 mM sodium acetate
(pH 4.5), 150 mM NaCl; 2 mM EDTA);
[0114] (8) Refolded protein is stored at 4.degree. C. for 1-48
hours before chromatographic purification;
[0115] (9) Liquid chromatographic purification is begun by 0.16
.mu.m tangential filtration. The chemokine is captured by
chromatography using a strong cation column;
[0116] (10) Column is washed in buffer (40 mM Na acetate (pH 6.0),
250 mM NaCl);
[0117] (11) Elution is carried out using a step wise gradient
consisting of 500, 1000, & 1500 mM NaCl (biologically active
chemokines are found in the 500 and 1000 mM NaCl steps);
[0118] (12) Chemokine fractions are diluted 2-4 times in buffer
(appropriate for both anion and cation chromatography) followed by
tandem chromatography using strong anion and weak cation exchange
chromatography;
[0119] (13) The weak cation column is washed in buffer (40 mM Na
acetate (pH 6.0), 150-250 mM NaCl); and
[0120] (14) Chemokine is eluted from the weak cation column with a
10-20 column volume linear gradient of 250-1000 mM NaCl ; and
[0121] (15) The peak fractions are further polished through a size
exclusion column (sephacryl S-100) equilibrated with 50 mM NaOAc at
pH 6, 150-300 mM NaCl.
[0122] The eluted fractions obtained from step (15) are analyzed
using SDS-PAGE and the chemokine containing peak fractions are
combined.
[0123] Using the process described above, 50% purity can be
achieved after the initial recovery and isolation of the inclusion
bodies. The refolded chemokines are about 60-70% pure with a
greater than 60% refolding efficiency. The subsequent
chromatographic steps yield chemokine proteins (e.g., MPIF-1) that
are typically of greater than 95% purity. As noted in Example 1, no
major contaminant bands were visualized by silver staining when 40
.mu.g of MPIF-1 purified by this method was analyzed by SDS-PAGE.
(FIG. 9)
[0124] As noted above, the skilled artisan will recognize that
additional purification steps may be performed on the isolated
chemokine using techniques that are well known to those skilled in
the art.
[0125] The method of the present invention provides a very unique
process for purifying proteins from inclusion bodies. The process
itself is very simple, scalable and reproducible for all seven
chemokines tested (i.e., MPIF-1, MPIF-1d23, MIP-1.alpha., M-CIF,
MIP-4, Ck-.beta.-13, Ck-.alpha.-4).
[0126] The invented process greatly improves the yield of the
exemplified recombinant chemokines from cell (e.g., bacterial)
lysates. Further, the resulting protein is highly homogenous and
biologically active.
[0127] The process of the present invention has been shown to be
very effective in removal of endotoxin. Endotoxin removal is a very
important part of protein purification, especially for proteins
produced in E. coli that will be used therapeutically. The method
of the present invention can separate the target protein away from
greater than 90% of endotoxin contamination at the very first step.
The final purified protein contains 100 million-fold less endotoxin
(i.e., 0.01-1 EU/mg).
[0128] This method can be applied to the production of other
chemokines which form inclusion bodies when expressed in host cells
and has resulted in an increase of production scale from several
milligrams to grams for MPIF-1, MPIF-1d23, MIP-1.alpha., M-CIF,
MIP-4, Ck-.beta.-13, and Ck-.alpha.-4. As a result, large
quantities of such products can be cGMP manufactured for FDA IND
submission and Phase I Clinical Trials using the methods of the
present invention.
[0129] Process for the Purification of Secreted Target Proteins
[0130] Another aspect of the invention provides for the
purification of target proteins that have been expressed in a
secreted form from insect cells and mammalian cells. Since the
secreted target proteins expressed in these systems are not in the
form of inclusion bodies, the protein would not require
solubilization. The process of the invention for the purification
of secreted proteins is as follows.
[0131] The target protein samples are clarified through a 0.16
.mu.m tangential filtration unit. The filtered sample is then
captured through a strong cation exchange column. Suitable column
materials are well known to those skilled in the art. Examples of
strong anion exchange column materials include POROS HS-50 column,
S Sepharose, SP Sepharose, Reseource/Source S, Toyopearl S. The
cation column is washed with a buffer containing 40 mM sodium
acetate pH 4.5 to pH 6.0, 250 mM sodium chloride, eluted with steps
of 500 mM, 1000 mM, 1500 mM sodium chloride. Target protein is
eluted at the 500-1000 mM salt steps.
[0132] The cation column purified samples are diluted 4-fold, then
applied onto a strong anion/weak cation tandem columns. Suitable
such column materials are well known to those skilled in the art.
Examples of such columns include POROS HQ-50/POROS CM-20.
Alternative materials for the strong anion column for the tandem
column include Mono Q, Q Sepharose, Resource/Source Q, DEAE,
Toyopearl Q. Alternative materials for the weak cation column for
the tandem column include CM Sepharose, Toyopearl CM. The target
protein will bind to either the anion or to the cation column
depending upon the particular protein. As will be understood by
those skilled in the art, the arrangement of the column materials
in the tandem column chromatography step can be reversed depending
on which type of column material will bind target protein. The
tandem columns are washed with 40 mM sodium acetate pH 4.75-6.0,
150-250 mM sodium chloride. The column having the bound target
protein is eluted with a linear gradient of 250-1000 mM NaCl in
10-20 column volumes. The eluted fractions are analyzed through
SDS-PAGE and the target protein containing peak fractions are
combined.
[0133] As the skilled artisan will recognize, additional
purification steps may be performed on isolated target protein
using techniques that are well known to those skilled in the art.
For example, additional chromatography steps such as size
exclusion, ion exchange, hydrophobic interaction, affinity, reverse
phase may be employed.
[0134] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
EXAMPLES
Example 1
[0135] Purification of MPIF-1d23 from E. coli
[0136] A bacterial expression construct with the chemokine gene
without the secreted signal peptide was made and used to transform
E. coli host cells. IPTG induction of the lac Z promoter is used to
express the chemokine. Other induction modes can also be used.
[0137] Recent studies have shown that some N-terminus truncated
MPIF-1 variants have higher biological efficacy compared to the
full length protein. Many forms of MPIF-1 variants were constructed
and subsequently expressed in E. coli. and purified.
[0138] MPIF-1d23 is a variant in which 23 residues are deleted from
the N-terminus of MPIF-1. MPIF-1d23 is produced in E. coli with
IPTG induction and is present in the insoluble fraction (so called
inclusion body) after cell lysis.
[0139] Methods
[0140] Expression of MPIF-1d23
[0141] The MPIF-1d23 gene was inserted into the expression vector
pQE7 by using recombinant DNA techniques well known to those
skilled in the art. The coding sequence of the MPIF-1d23 was
amplified by PCR during which unique restriction sites,
SphI/HindIII, were introduced thereby allowing the gene to be
cloned into the E.coli expression vector pQE7. The resulted plasmid
DNA was used to transform E. coli M15 Rep4 host cell. For small
scale fermentation, bacterial transformant was grown in LB medium
containing Ampicillin and Kanamycin, then induced for production of
chemokine with 1 mM IPTG for 3 hours. For large scale production, a
semi defined medium or defined medium without antibiotics was
used.
[0142] Solubilization of MPIF-1d23 from E. coli
[0143] MPIF-1d23 was produced as insoluble protein deposited as
inclusion bodies. The method of MPIF-1d23 production utilizes a
series of solubilization procedures to separate the target protein
from the host contaminants by: cell lysis, 0.5M NaCl washes, to
isolate inclusion body that containing partially purified
MPIF-1d23. In details, E. coli cell paste was resuspended in a
buffer containing 100 mM Tris pH 7.4, 25 mM EDTA, passed through a
Microfluidizer twice at 6000-8000 psi. The lysed sample was mixed
with NaCl to a final concentration of 0.5M, then spun at
7000.times.g for 15 minutes. The resulting pellet was washed with
the same buffer plus 0.5M NaCl again as partially purified
inclusion body.
[0144] The partially purified inclusion body was subjected to
further solubilization with 1.5-1.75 M guanidine hydrochloride for
2-4 hours at 4-25.degree. C. MPIF-1d23 was soluble and remained in
the soluble phase after 7,000.times.g centrifugation. This sample
was placed at 4.degree. C. for further solubilization overnight,
then centrifuged at 30,000.times.g. The supernatant is called a
1.75M GuHCl extract of which 50-70% is MPIF-1d23 protein.
[0145] Refolding
[0146] The 1.75M GuHCl extracted chemokine was refolded by quickly
mixing the GuHCl extract with 10 volumes of a refold buffer
containing 50 mM sodium acetate pH 4.5, 125 mM sodium chloride, 2
mM EDTA with 30 minutes of vigorously stirring. The mixture was set
at 4.degree. C. without mixing for 0.5-48 hours prior to the
chromatographic purification steps described below.
[0147] Liquid Chromatographic Purification of MPIF-1
[0148] The refolded MPIF-1d23 samples were clarified through a 0.16
.mu.m tangential filtration unit. The filtered sample was captured
through a strong cation exchange (poros HS-50) column. The HS-50
column was washed with a buffer containing 50 mM sodium acetate pH
6.0, 300 mM sodium chloride, eluted with steps of 500 mM, 750 mM,
1000 mM, 1500 mM sodium chloride.
[0149] The HS-50 0.5 M sodium chloride eluted fraction was diluted
2-fold, then applied onto a set of anion (poros HQ-50) and cation
(poros CM-20) exchange columns in a tandem mode. Both columns were
washed with 50 mM sodium acetate pH 6.0, 150 mM sodium chloride.
The CM column was eluted with a linear gradient of 150-750 mM NaCl
in 10-20 column volumes. Elute fractions were analyzed through
SDS-PAGE and RP-HPLC. The MPIF-1d23 containing peak fractions which
has the expected RP-HPLC profile were combined. The HQ/CM purified
MPIF-1d23 was further polished by a size exclusion (Sephacryl
S-100) chromatographic step.
[0150] Purification of MPIF-1 and MPIF-1d23
[0151] A production process of MPIF-1 described in the method
section above was used to purified MPIF-1d23. A 50-70% purity can
be achieved after the initial recovery and isolation of inclusion
body. From the 1.75 M GuHCl extract, the refolding efficacy of
MPIF-1d23 is greater than 80%.
[0152] Refolded sample was subsequently purified through three
chromatographic steps. After the HS-50 and HQ/CM steps, MPIF-1d23
sample is typically 98% pure. The recovery of the chromatographic
procedures is greater than 50%. Only two minor bands (approximately
50 ng each) are seen by silver staining when 40 .mu.g of such
purified protein is loaded. Size exclusion chromatography as the
last step was very effective in removing these two minor
contaminants. This step also will be useful as a buffer exchange
step for final bulk formulation of the protein. The final purified
MPIF-1d23 is free of any visible contaminating bands by silver
staining when 40 .mu.g of protein is loaded. (FIG. 9).
[0153] Table I is an outline example of the purification results.
FIG. 1 shows the SDS-PAGE of the purified protein. Typically, the
yield of MPIF-1 is 1 gram per kilogram E coli cell paste.
1TABLE 1 Purification Table of MPIF-1d23 Purified Total Protein
Total Estimated MPIF- Re- Volume Conc. Protein Purity 1d23 covery
Step (ml) (mg/ml) (mg) (%) (mg) (%) E. coli (24000) (1.2 Kg.
Culture Wt.) GuHCl 6000 5 3000 60% 1800 100 Extract 0.16 .mu.m
50000 0.05 2500 70% 1750 97 filtration HS pool 650 2.5 1625 90%
1462 81 HQ/CM pool 140 8.2 1148 98% 1125 63 S-100 pool 275 4.0 1100
99% 1089 61
[0154] Analysis of Purified MPIF-1
[0155] 1. Size Exclusion Chromatography
[0156] A monomeric MPIF-1d23 is 8.9 kD, and a dimer will be 17.8
kD. Purified MPIF-1d23 runs as a single symmetrical peak just
behind the 17 kD (myoglobin) molecular marker. Because of the
limited resolution of the sizing column at this molecular weight
range, it cannot be precisely determined if the protein is a
monomer. However, mass spec data and primary NMR analysis indicate
that purified MPIF-1d23 is monomeric.
[0157] 2. Reverse Phase HPLC
[0158] Active MPIF-1d23 purified from E. coli as described above
has a retention time of 6.15 (.+-.0.1) minutes with a shoulder at
6.8 (.+-.0.1) minutes in a C8 RP-HPLC analysis while other isoforms
have longer retention time such as 6.7 to 7.1 minutes. The active
MPIF-1d23 reversed phase HPLC profile is very similar to those of
the MPIF-1 purified from baculovirus infected Sf-9 supernatant and
CHO cell supernatant.
[0159] It has not yet been fully determined what component
corresponds to the shoulder peak. Since this main peak plus
shoulder profile is also seen in baculovirus (HG00300-B5,
HG00300-B7) and CHO (HG00311-C1) expressed MPIF-1 which does not
involve a refolding process, the shoulder peak is not likely
generated from incomplete refolding. Mass spec analysis show that
the main peak and shoulder have the exact same molecular weight as
expected from the amino acid sequence of MPIF-1d23.
[0160] 3. Endotoxin Level
[0161] Purified MPIF-1 from the above process contains low level of
endotoxin, typically below 0.1 EU/mg purified protein.
[0162] 4. IEF
[0163] Final purified MPIF-1 runs as a single band on an
isoelectric focusing gel. This is indicative of homogeneity of the
protein.
[0164] Characterization of MPIF-1d23 Isoforms
[0165] We have observed the presence of different isoforms of
MPIF-1d23 from E. coli cell paste. With the purification methods
described above, many different active isoforms MPIF-1d23 were
isolated
[0166] When MPIF-1d23 inclusion body pellet was extracted with 1.75
M GuHCl for 2-4 hours, normally 50% of MPIF-1d23 is in the soluble
phase. The other 50% of MPIF-1d23 which is not solubilized under
such conditions can be extracted out by higher concentration of
GuHCl, then refolded, purified and tested for HPLC profiles and
bioactivities. It was found that such populations of MPIF-1d23 have
different CM and RP-HPLC profiles, and they are less active or
inactive in our in vitro bioassays. Therefore, there are different
isoforms of MPIF-1d23 present in the inclusion body. By
solubilizing inclusion body with low concentration GuHCl, the
primarily active portion of MPIF-1d23 can be selectively extracted
out from a heterogeneous MPIF-1d23 population.
[0167] The 1.75M GuHCl extract was refolded through dilution.
Greater than 80% of MPIF-1d23 remains in solution and refolded
protein is 60-70% pure as analyzed by SDS-PAGE. Such sample was
captured in a HS column and eluted using NaCl steps into 0.5 M,
0.75M, 1.0M and 1.5 M sodium chloride fractions. Each of the
fraction contains MPIF-1d23 protein and shows same protein band on
SDS-PAGE. Our analysis on each fraction shows that the 0.5 M sodium
chloride fraction contains the most active MPIF-1d23 while other
fractions eluted with higher concentrations of sodium chloride have
different CM and RP-HPLC profiles than the 0.5M fraction and
subsequently are less or not active in the bioassay. Therefore, the
HS step elution further separate the active MPIF-1d23 from other
less active isoforms.
[0168] The HS purified MPIF-1d23 was subsequently applied onto a
set of HQ-50 and CM-20 columns in a tandem mode. MPIF-1d23 was
eluted from CM column with a linear gradient as a main peak at
approximately 400 mM sodium chloride in 50 mM sodium acetate pH
6.0. Several small peaks are often present after the main peak and
they were identified as the less active isoforms by RP-HPLC and
bioassays (see Table 2).
2TABLE 2 Comparison of Different CM Peak of MPIF-1d23 RP-HPLC
Sample Retention Ca++ LPP-CFC MPIF-1d23 Elution Time Mobilization
Inhibition Sample (mM NaCl) (min) (ng/ml)* (ng/ml)# CM Peak 1 400
mM 6.1 100 1 CM Peak 2 450 mM 6.1, 6.71 100 100 CM Peak 3 500 mM
6.8 1000 >1000 *Minimum concentration required to mobilize
calcium in THP-I cells #Concentration producing 50% inhibition of
LPP-CFC colonly formation compared to the control
[0169] The three CM peaks have identical sizing HPLC profiles. Mass
Spec analysis shows that the CM peak 1 and peak 3 samples have the
exact same molecular mass as expected for a monomeric MPIF-1d23
based on its amino acid sequence. Since MPIF-1d23 has 6 cysteins,
there could be many different disulfide bond related conformations.
To demonstrate this possibility, the two MPIF-1d23 fractions were
treated with 6M GuHCl with or without 50 mM DTT at room temperature
overnight, then refolded and purified using the same methods
described above. Samples treated with 6 M GuHCl without DTT retain
their original RP-HPLC profiles. However, peak 3 sample treated
with 6 M GuHCl plus 50 mM DTT presented a shift of RP-HPLC profiles
which contains 50% of the 6.1 minute peak. There is a possibility
that these two isoforms have different disulfide linkages. With
treatment of GuHCl plus DTT, a less active isoform was converted to
active MPIF-1d23 through rearrangement of the disulfide
linkage.
[0170] Discussion
[0171] The foregoing demonstrates that the purification method of
the present invention results in the successful recovery and
purification of E. coli derived chemokines. The advantages of using
this method are that it is simple, it can be done at relatively low
cost and, thus, can be easily scaled up and adapted for cGMP
manufacturing. It yields a biologically active, highly homogeneous
product with no aggregated forms of the target protein, and good
recovery of chemokine is achieved with high purity and extremely
low endotoxin level.
[0172] This method can be applied to the production of other
chemokines from E. coli and has resulted in an increase of protein
production scale for MPIF-1, MPIF-1d23, MIP.alpha.1, M-CIF and
MIP-4, among others, from several milligrams to gram quantities. As
a result, large quantities of target proteins can be cGMP
manufactured to achieve goals for IND submissions and Phase I
Clinical Trials.
Example 2
[0173] GuHCl Titration
[0174] Since the GuHCl concentration for inclusion body
solubilization might play a role in the initial output of the
active and inactive MPIF-1d23, a GuHCl titration experiment was
performed to optimize the GuHCl extraction step and systematically
evaluate the process. MPIF-1d23 inclusion body was prepared as
described, then solubilized with 0.75M, 1.5M, 2.0M, 3.0M, 4.0M, and
6.0M GuHCl for three hours.
[0175] The initial extraction shows that 0.75M GuHCl was able to
extract out some MPIF-1d23, 1.5-2.0M GuHCl has better extraction,
3.0M to 6.0M GuHCl, considerable amount of other contaminants are
present in the extract (see FIG. 10). After refolding through
dilution to final concentration of 150 mM GuHCl, samples were
captured in a HS column and eluted with steps of 0.3 M, 0.5M,
0.75M, 1.0 M and 1.5M NaCl. Previous experiments showed that active
MPIF-1d23 was eluted in the 0.5M NaCl step. 0.5M NaCl pooled
MPIF-1d23 was further purified through HQ/CM tandem columns and the
first peak which was identified as the active MPIF-1d23 peak from
CM column was pooled as the final purified sample.
[0176] The purified samples were analyzed by SDS-PAGE, RP-HPLC,
total yield, and calcium mobilization activity (Table 3). Table 3
shows that 1.5M to 2.0M GuHCl extraction produced the best recovery
and best purity for active MPIF-1d23. However, 0.75M GuHCl is not
enough to dissociate MPIF-1d23 from the inclusion body. While high
concentration of GuHCl seems to extract out more MPIF-1d23
initially as well as other contaminants from inclusion body as
shown on SDS-PAGE (FIG. 10), this MPIF-1d23 band includes the
non-active MPIF-1d23 isoform. The presence of these contaminants
might significantly interfere with the refolding. The final result
is less active protein and lower purity from higher concentration
of GuHCl extraction. Therefore, the range of about 1.5M to about
1.75M GuHCl appears to be optimal.
3TABLE 3 Summary of GuHC1 Titration Experiments Total Purity by
RP-HPLC# Ca++ Recovery SDS-PAGE Active Peak Mobilization* Sample
(mg) (%) (%) (nM) 0.75M. GuHCl 0.33 mg 80% 38% 124,224 1.5M GuHCl
17.82 mg 98% 80% 159,253 2.0M GuHCl 25.74 mg 98% 75% 126,262 3.0M
GuHCl 11.76 mg 95% 73% 141,268 4.0M GuHCl 4.29 mg 92% 65% 113,245
6.0M GuHCl 3.3 mg 90% 37% 109,208 *change in intracellular calcium
at 100 and 1000 ng/ml of MPIF-1d23, respectively #Percentage of the
6.1 minutes peak integrated area to the overall peak area.
Example 3
[0177] Refolding Time for MPIF-1d23
[0178] To optimize the refolding process, a refold time course was
performed. The 1.75 GuHCl extract of MPIF-1d23 was prepared as
described in the method above, then diluted 10-fold in the
refolding buffer (50 mM sodium acetate pH 4.5, 125 mM sodium
chloride, 2 mM EDTA). Refolded samples were taken after one, three,
six and twenty hours. Each time course sample was processed through
the HS and HQ/CM chromatographic steps.
[0179] The purified samples were analyzed by SDS-PAGE, RP-HPLC,
total yield, and calcium mobilization activity. Table 4 shows that
sample taken just one hour after dilution is just as active and
pure as the one taken after 20 hours refold. Therefore, there is no
need for lengthy refolding in our process. Perhaps, a portion of
MPIF-1d23 present in the inclusion body population is already in a
corrected folded conformation. This portion of protein can be
easily dissociated from inclusion body by low concentration of
GuHCl which does not cause major denaturation, therefore, no major
refolding process is needed.
4TABLE 4 Summary of Refolding Time Course Experiments Total Purity
by RP-HPLC# Ca++ Recovery SDS-PAGE Active Peak Mobilization* Sample
(mg) (%) (%) (nM) Refold TC 1 4.8 98% 80% 160, 259 hour Refold TC 3
4.5 98% 80% 119, 257 hours Refold TC 6 4.4 98% 80% 136, 273 hours
Refold TC 20 5.1 98% 80% 149, 250 hours *Change in intracellular
calcium at 100 and 1000 ng/ml of MPIF-1d23, respectively.
#Percentage of the 6.1 minutes peak integrated area to the overall
peak area.
Example 4
[0180] Endotoxin Removal
[0181] Endotoxin removal is an important step of protein
purification, especially in the case of E. coli derived product.
During chemokine purification process, the level of endotoxin at
different steps was monitored and found that over 90% of endotoxin
can be removed through the step of inclusion body isolation (see
Table 5). The final purified MPIF-1d23 contains an extremely low
level of endotoxin.
5TABLE 5 Endotoxin Removal During the Chemokine Purification
Process Vol Sample EU/ml (ml) Total EU % E. coli cell paste 5
.times. 10.sup.5 2000 1.0 .times. 10.sup.9 100 Cell lystate supern
6 .times. 10.sup.5 2000 1.2 .times. 10.sup.9 120 Cell lystate
pellet 4.4 .times. 10.sup.4 2000 8.8 .times. 10.sup.7 5.4 Refold
0.26 5500 1.5 .times. 10.sup.3 0.00015 HS pool 2.4 550 1.3 .times.
10.sup.3 0.00013 HQ/CM pool 1 0.6 60 36 0.0000036 HQ/CM pool 2 0.09
60 5.4 0.00000054
Example 5
[0182] Purification: of M-CIF from E. coli
[0183] The M-CIF gene was inserted into the expression vector pQE6
using recombinant DNA techniques well known to those of skill in
the art. Briefly, the coding sequence of M-CIF was amplified by PCR
during which unique restriction sites (BsphI and BamHI) were
introduced thereby allowing the gene to be cloned into the E. coli
expression vector pQE60. The resulting plasmid DNA was used to
transform E. coli M15 Rep4 host cells. Cell cultures were prepared
as described in Example 1.
[0184] The purification involves the following steps, and unless
otherwise specified, all procedures were conducted at 4-10.degree.
C.
[0185] M-CIF was produced as insoluble protein deposited in
inclusion bodies. Upon completion of the production phase of the E.
coli fermentation, the cell culture was cooled to 4-10.degree. C.
and the cells were harvested by continuous centrifugation at 15,000
rpm (Heraeus Sepatech). On the basis of the expected yield of
protein per unit weight of cell paste and the amount of purified
protein required, an appropriate amount of cell paste, by weight,
was suspended in a buffer solution containing 100 mM Tris, 50 mM
EDTA, pH 7.4 in a 10-20 ml buffer per gram cell paste ratio. The
cells were dispersed to a homogeneous solution using a high shear
mixer.
[0186] The cells were then lysed by passing the solution through
microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at
4000-12000 psi. The homogenate was mixed with a NaCl solution to a
final concentration of 0.5 M NaCl, followed by centrifugation at
7,000.times.g for 15 min. The resulting pellet was washed again
using 0.5 M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4, followed by
centrifugation at 7000.times.g for 15 min.
[0187] The washed inclusion bodies pellet) were solubilized with
1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After
centrifugation at 7,000.times.g for 15 min., the pellet was
discarded and the M-CIF-containing supernatant was placed at
2-10.degree. C. overnight.
[0188] Following high speed centrifugation (30,000.times.g) to
remove the insoluble particles, the GuHCl solubilized proteins were
refolded by vigorously stirring the GuHCl extract with 20 volumes
of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA.
The refolded diluted protein solution was kept at 2-10.degree. C.
without mixing for 1-72 hours prior to further purification
steps.
[0189] To clarify the refolded M-CIF solution, a previously
prepared tangential filtration unit equipped with 0.16 .mu.m
membrane filter with appropriate surface area (Filtron),
equilibrated with 40 mM sodium acetate, pH 6.0 was employed. The
filtered sample was loaded onto a cation exchange of POROS HS-50
resin (Perseptive Biosystems). The column was washed with 40 mM
sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and
1500 mM NaCl in the same buffer, in a stepwise manner. The
absorbance at 260 nm and 280 nm of the effluent was continuously
monitored. Fractions were collected and fuirther analyzed by
SDS-PAGE.
[0190] Those fractions containing the desired protein were then
pooled and mixed with 4 volumes of water. The diluted sample was
then loaded onto a previously prepared set of tandem columns of
strong anion (POROS HQ-50, Perseptive Biosystems) and weak cation
(POROS CM-20, Perseptive Biosystems) exchange resin. The columns
were equilibrated with 40 mM sodium acetate, pH 6.0. Both columns
were washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The
CM-20 column was then eluted using a 10 column volume linear
gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to
1.0 M NaCl, 50 mM sodium acetate, pH 6.0. Fractions were collected
under constant A280 monitoring of the effluent. Those fractions
containing the protein of interest (determined by SDS-PAGE) were
then pooled.
[0191] The resultant M-CIF was of greater than 95% purity after the
above refolding and purification steps. No major contaminant bands
were observed from the Coomassie Blue stained 16% SDS-PAGE gel when
5 .mu.g of purified protein was loaded. The purified protein was
also tested for endotoxin/LPS contamination. The LPS content was
less than 0.1 ng/ml according to LAL assays.
Example 6
[0192] Purification of FGF-13 from E. coli
[0193] The coding sequence of FGF-13 was amplified by PCR during
which unique restriction sites SphI and HindIII were introduced and
then cloned into the E. coli expression vector pQE7. The resulting
plasmid DNA was used to transform E. coli M15 Rep4 host cells. Cell
cultures were prepared as described in Example 1. FGF-13 (HODAH63)
protein, was produced in E. coli transformant as two bands in
modest amount with IPTG induction.
[0194] The purification involves the following steps, and unless
otherwise specified, all procedures were conducted at 4-10.degree.
C.
[0195] Solubilization of FGF-13 from E. coli
[0196] FGF-13 was produced as insoluble protein deposited in
inclusion bodies. Upon completion of the production phase the E.
coli fermentation, the cell culture was cooled to 2-10.degree. C.
and the cells were harvested by continuous centrifugation at 15,000
rpm (Heraeus Sepatech). Per gram of E. coli cell paste was
resuspended in 10 ml buffer containing 100 mM Tris-HCl pH 7.5, 50
mM EDTA using a shear mixer.
[0197] The cells were lyzed in a microfluidizer (Microfluidics,
Corp. or APV Gaulin, Inc.) at 4,000-12,000 psi twice. The
homogenate was mixed with a NaCl solution to a final concentration
of 0.5 M NaCl, followed by centrifugation at 7,000.times.g for 15
minutes. The resulting pellet was washed again using 100 mM
Tris-HCl pH 7.4, 50 mM EDTA, 0.5 M NaCl, followed by centrifugation
at 7000.times.g for 15 min.
[0198] The washed inclusion bodies (pellet) were extracted with 2 M
GuHCl in 50 mM Tris pH7.5, 25 mM EDTA for 2-4 hours. After
centrifugation at 7000.times.g for 15 minutes, the FGF-13
containing supernatant was placed at 2-10.degree. C. overnight
followed by centrifugation at 30,000.times.g for 20 minutes. This
supernatant which contains 60-80% FGF-13 is called the 2 M GuHCl
extract.
[0199] Refolding
[0200] The 2M GuHCl extract was refolded by quickly mixing the
GuHCl extract by vigorously stirring for 30 minutes with 20 volumes
of a buffer containing 50 mM Tris pH7.5, 25 mM EDTA, 200 mM NaCl,
20 .mu.g/ml Pefabloc SC, 2 .mu.g/ml E-64 (Boehringer Mannheim). The
mixture was placed at 2-10.degree. C. without mixing for 1-48
hours.
[0201] Liquid Chromatographic Purification of FGF-13
[0202] The refolded FGF-13 was clarified through a 0.16 .mu.m
tangential filtration unit. The filtered sample was applied to a
POROS HS-50 (PerSeptive Biosystem) cation exchange column at pH7.5.
The HS column was washed with a buffer containing 50 mM Tris pH
7.5, 0.5M NaCl, and eluted with 750 mM, 1.2 M, and 1.5 M NaCl
steps. FGF-13 was eluted in the 1.5 M NaCl step.
[0203] The HS purified FGF-13 was diluted 4-fold, then applied onto
a set of POROS HQ-50/POROS CM-20 (PerSeptive Biosystem)
anion/cation columns in a tandem chromatographic mode. Both columns
were washed with a buffer containing 50 mM Tris pH 7.5, 400 mM
NaCl. The CM column was eluted with a 20 column volume of 0.4 M to
1.25 M NaCl linear gradient. FGF-13 was eluted with approximately
700 mM NaCl, and finished with a S200 Sepharcryl HR (Pharmacia)
size exclusion column at greater than 95% purity.
[0204] Results
[0205] FGF-13 transformed E. coli produced two bands after IPTG
induction. After cell lysis, FGF-13 protein was extracted with
GuHCl from the insoluble fraction as described in the method
section. The GuHCl extracted protein appears to be the same size as
the starting material on SDS-PAGE and is greater than 60% pure.
However, two additional minor bands which seem to be 2 kD smaller
than the original two bands appeared on SDS-PAGE after refolding.
Proteolytic degradation might occur during refolding and four bands
are present.
[0206] Refolded FGF-13 was captured by a strong cation exchange
POROS HS-50 column and eluted at 90% purity with 1.25 to 1.5M NaCl.
The resulted protein was passed through a set of tandem columns
consisting of a strong anion exchange (POROS HQ-50) column and a
weak cation exchange (POROS CM-20). FGF-13 was eluted from the CM
column with 700-800 mM NaCl. At least four different bands at MW
18-22 kD appear on reduced SDS-PAGE in the CM purified FGF-13 (FIG.
10). All four bands are confirmed to be FGF-13 protein by
N-terminal sequence analysis.
6 The upper 1st band: MQGEN (SEQ ID NO: 1) the expected N- terminus
The upper 2nd band: MQGEN (SEQ ID NO: 1) the expected N- terminus
3rd and 4th bands: TDQLS (SEQ ID NO: 2) 21 residues shorter than
above
[0207] From the above results, it was expected that the C-terminal
degradation might occur to the 2nd and the 4th bands. The cleavage
site was determined based on mass through Mass Spec analysis. It
was concluded that 9 amino acid residues were removed at the
C-terminus from a small percentage of the protein.
7 Full length sequence of FGF-13 75% 20% MQGEN (SEQ TDQLS (SED ID
ID NO:1) NO:2) :::: ::::: MGAARLLPNL TLCLQLLILC CQTQGENHPS
PNFNQYVRDQ GAMTDOLSRR QIREYQLYSR TSGKHVQVPG RRISATAEDG NKFAKLIVET
DTFGSRVRIK GAESEKYICM NKRGKLIGKP SGKSKDCVFT EIVLENNYTA FQNARHEGWFMV
FTRQGRPRQA SRSRQNQREA HFIKRLYQGQ LPFPNHAEKQ KQFEFVGSAP TRRTK RTRRP
OLPT (SEQ ID NO:3)
Example 7
[0208] Biological Activity of MPIF-1 Purified by the Inventive
Process
[0209] MPIF-1 protein has been expressed in baculovirus, E. coli
and CHO expression systems and purified to >95% homogeneity as
determined by Coomassie Blue staining of an SDS-PAGE gel. The
mammalian expressed and purified protein was composed of 99 amino
acids with an RVTKDAE (SEQ ID NO: 4) amino acid sequence at the
N-terminus and contained neither mannose nor N-acetylglucosamine,
as expected from the absence of consensus N-linked glycosylation
sites in the deduced primary amino acid sequence. Purified MPIF-1
from E.coli has an additional methionine followed by the "RVTKDAE"
MPIF-1 sequence at the N-terminus. Purified MPIF-1 from
baculovirus, E. coli and CHO expression systems have been found to
stimulate chemotaxis of T-lymphocytes and granulocytes in vitro,
induce a transient rise in the intracellular calcium concentration
in monocytes, and inhibit colony formation by mouse bone marrow
derived Low Proliferative Potential Colony-Forming Cells (LPP-CFC)
in vitro.
[0210] To demonstrate that MPIF-1 purified according to the process
of the present invention is biologically active, the following in
vitro and in vivo experiments were performed.
[0211] Materials and Methods
[0212] Reagents and chemicals: Hanks Balanced Salt Solution (HBSS),
IMDM, and MEM tissue culture media were purchased from Life
Technologies Inc., Gaithersburg, Md. Fetal bovine serum (FBS),
Histopaque 1077, and Histopaque 1119 were purchased from Sigma
Tissue Culture Products, St. Louis, Mo. MyeloCult.TM. H5100 and
MethoCult.TM. H4535 growth medium for culturing hematopoietic
progenitors were purchased from Stem Cell Technologies Inc.,
Vancouver, BC, Canada. Human and murine recombinant cytokines were
all purchased from R and D Systems Inc., Minneapolis, Minn.
QBEND/10 CD34 cell isolation kit, Type RS columns, and VarioMac
were purchased from Miltenyi Biotech Inc., Sunnyvale, Calif. All
the monoclonal antibody reagents against the human hematopoietic
cell surface antigens were purchased from Becton Dickinson
Immunocytometry Systems, San Jose, Calif.
[0213] Mouse bone marrow cells: Mouse bone marrow cells (MBMC) were
isolated from the femur and tibia of 6-8 week old, female, C57B1/6
mice (Jackson Laboratories, Barharbor, Mass.) by flushing with IMDM
supplemented with 5% FBS. A low density fraction of the cell
population was then obtained by centrifuging (750.times.g for 30
min. at 23.degree. C.) the cell suspension over a cushion of
Histopaque (1.119 gm/ml). Cells sedimenting at the interface of the
medium and the ficoll were washed three times with IMDM growth
medium, plated in a 10 cm diameter tissue culture dish (Costar,
Cambridge, Mass.), and then incubated for 1 hour at 37.degree. C.
in a tissue culture incubator to remove cells capable of adhering
to the treated polystyrene surface. The resulting non-adherent
populations of cells were then used as target cells in clonogenic
assays. For some experiments, MBMC's were enriched by negative
immunoselection for primitive hematopoietic progenitors. Briefly, a
low density fraction of MBMC's were treated at 4-8.degree. C. with
a panel of rat monoclonal antibodies against murine antigens
(CD11b, CD4, CD8, CD45R, and Gr. 1). Antibody-bound, committed
hematopoietic precursors and mature cells were then removed by
incubation with immunomagnetic beads. The resulting populations of
cells, referred to as lineage-depleted populations of cells
(Lin-cells), is typically 60- to 80-fold enriched for High
Proliferative Potential Colony-Forming Cells (HPP-CFC) as
determined by clonogenic assays (see below).
[0214] CD34.sup.+ human hematopoietic stem cells: CD34.sup.+ cells
were isolated from human cord blood by immunoselection using
QBEND/10 Cell Isolation Kit and high-gradient magnetic cell sorting
according to the manufacturers instructions. Briefly, 45 ml of cord
blood was collected in 5 mM EDTA solution with 20 U/ml heparin and
centrifuged at 200.times.g for 10 minutes at 23.degree. C. The
buffycoat was then resuspended in PBS supplemented with 5 mM EDTA
and centrifuged on Histopaque (density 1.077 g/ml). PBMCs were
collected by harvesting the interface band and washed three times
with PBS containing 5 mM EDTA, resuspended in ice cold PBS with 5
mM EDTA and 0.5% BSA, and then subjected to the QBEND/10 Cell
Isolation protocol. Typically, this procedure yielded a population
of cells that was 95% CD34.sup.+ as determined by FACScan.
[0215] Clonogenic assays on mouse bone marrow cells: HPP-CFC and
LPP-CFC colony formation assays were performed in a two-layered
agar culture system. Briefly, the bottom layer was prepared in 3.5
cm diameter tissue culture dishes with 1 ml of MEM medium
supplemented with 20% FBS, 0.5% Difco agar, and a panel of
recombinant cytokines that consisted of mouse IL-3 (7.5 ng/ml),
mouse SCF (75 ng/ml), human M-CSF (7.5 ng/ml), and mouse IL-1 a (15
ng/ml). Chemokines were also incorporated into the bottom agar
where indicated. This layer was then overlayed with 0.5 ml of
murine bone marrow cell suspension (1,500 cells/dish) prepared in
the agar medium described above except that it contained 0.3% agar
and no cytokines. The dishes were then incubated for fourteen days
in a tissue culture incubator (37.degree. C., 88% N.sub.2, 5%
CO.sub.2, and 7% O.sub.2) and colonies were scored under an
inverted microscope.
[0216] The above combination of cytokines typically stimulates
colony formation by two classes of progenitors; HPP-CFC is a
primitive, multipotential progenitor that exhibits many properties
of hematopoietic stem cells and gives rise to a colony of >5 mm
in diameter, whereas LPP-CFC is a committed progenitor which
differentiates along the granulocyte and monocyte lineages and
gives rise to colonies that are <1 mm in diameter. In some
experiments the impact of MPIF-1 on the growth of BFU-E, CFU-E, and
CFU-GM was also determined. Bone marrow cells (5.times.10.sup.4
cells) were suspended in 1.0 ml of MethoCult semisolid medium in
3.5 cm diameter tissue culture dishes. For CFU-E assays, the above
medium was supplemented with recombinant human Epo at 3 U/ml and
the dishes were incubated as above for two days. After this two day
incubation, hemoglobinized colonies (4-16 cells/colony) were
identified by acid benzidine stain and scored under an inverted
microscope using bright-field optics. For BFU-E and CFU-GM assays,
the above medium was supplemented with recombinant mouse IL-3 (5
ng/ml), GM-CSF (10 ng/ml), and Epo (3 U/ml) and the dishes were
incubated for eleven days. Colonies were then scored as above using
an inverted microscope.
[0217] Animal studies: Twelve to fifteen weeks old, female, C57B1/6
mice purchased either from Jackson Laboratories or Harlan
Industries were utilized throughout all experiments reported here.
Mice were fed a standard diet and housed under standard conditions
of lighting, temperature, and air. Neutropenia was induced by
administering (I.P.) a single dose of freshly prepared 5-Fu
solution (in warm distilled water) at 150 mg/Kg body weight. In
some experiments, two doses of 5-Fu were administered at the above
dosage. Blood was collected from mice either from the tail vein or
periorbital sinus in tubes containing 20 U/ml sodium heparin and 5
mM disodium EDTA as anticoagulants. White blood cell (WBC) counts
in the peripheral blood was determined using a Coulter counter.
[0218] Results
[0219] MPIF-1 Inhibits Colony Formation by LPP-CFC
[0220] Clonogenic assays were performed, where a limiting number of
low density population of mouse bone marrow cells were plated in
methylcellulose- or agar containing growth medium supplemented with
appropriate combinations of cytokines to demonstrate the effect of
MPIF-1 purified according to the present invention on hematopoietic
cell differentiation. No colonies were detected, of any kind,
either in the absence of any added cytokines or in the presence of
MPIF-1 alone. However, the numbers of cytokine stimulated CFU-GM
and LPP-CFC colonies in the presence of MPIF-1 were decreased to
30% of those found in the control cultures (Table 6). The
inhibitory effect of MPIF-1 appeared to be specific, as MPIF-1 had
no effect on colony formation by CFU-E, BFU-E, and HPP-CFC (Table
6). MCP-4, a .beta.-chemokine which was also expressed and purified
by the process of the present invention as MPIF-1, had no effect on
colony formation by CFU-GM, CFU-E, BFU-E, LPP-CFC, and HPP-CFC
(Table 6). Thus, as anticipated, the inhibitory effect of MPIF-1
was specific to LPP-CFC and this effect is not due to a contaminant
copurified in the MPIF-1 preparation.
[0221] LPP-CFC colony formation was inhibited in a dose-dependent
manner in the presence of MPIF-1 purified according to the present
invention; compared to the control there was 50% reduction in the
number of colonies at 20 ng/ml MPIF-1 and 73% reduction at 80 ng/ml
(FIG. 14B). No further increase in the inhibition of LPP-CFC colony
formation was observed when this preparation of MPIF-1 was tested
up to 500 ng/ml (data not shown). As expected, M-CIF in the same
assay had no effect on either LPP-CFC or HPP-CFC (FIG. 14B) and
MPIF-1 had no effect on HPP-CFC (FIG. 14A). Thus, there exists a
small (.about.20%) but significant percentage of LPP-CFC's in the
bone marrow that appear to be not inhibited by MPIF-1. MIP-1.alpha.
has been shown to inhibit the growth of multipotential
hematopoietic progenitors. Also, MIP-1.alpha. is most homologous to
MPIF-1 within the .beta.-chemokine family. Therefore, MIP-1.alpha.
was tested but it had no effect on either LPP-CFC or HPP-CFC colony
formation, whereas MPIF-1, as shown above, inhibited LPP-CFC colony
formation (FIG. 15).
[0222] MPIF-1 Inhibits Proliferation of Human CD34.sup.+
Progenitors
[0223] The data presented above demonstrates that human MPIF-1
purified according to the present invention has biological activity
in regards to murine hematopoietic progenitor. To assess whether
human MPIF-1 purified according to the present invention has
biological activity in regards to human cells, CD34.sup.+
hematopoietic stem cells were isolated from human cord blood and
cultured for four days in the presence of IL-3 and SCF. The
resulting populations of cells, consisting of myeloid progenitors,
were then allowed to undergo proliferation and differentiation for
six additional days in the presence or absence of multiple
cytokines plus various concentrations of MPIF-1 purified according
to the present invention. As shown in FIG. 16, there was little
proliferation of cells either in the absence of cytokines or in the
presence of MPIF-1 alone. Addition of cytokines resulted in a
.about.40-fold increase in total cells and this cytokine stimulated
proliferation of cells was inhibited, in a dose-dependent manner,
by MPIF-1 (FIG. 16). Clonogenic assays also demonstrated that
CFU-GM induced colony formation was sensitive to MPIF-1, as shown
above with murine cells (Table 6). Thus, as anticipated, MPIF-1
purified according to the present invention inhibits growth of
myeloid progenitors of both murine and human origin.
[0224] MPIF-1 Protects Myeloid Progenitors from 5-Fu-Induced
Cytotoxicity In Vitro
[0225] To demonstrate that the inhibitory effect of MPIF-1 purified
according to the present invention can lead to the protection of
LPP-CFC from the cytotoxicity of the cell cycle acting
chemotherapeutic drug 5-Fu, lineage-depleted populations of cells
(Lin.sup.- cells) were isolated from mouse bone marrow and
incubated in the presence of multiple cytokines with or without
MPIF-1. After 48 hours, one set of each culture received 5-Fu and
the incubation was then continued for an additional 24 hours. After
the 24 hour incubation, the numbers of surviving LPP-CFC were
determined by a clonogenic assay. As shown in FIG. 17, .about.40%
of LPP-CFC were protected in the presence of MPIF-1 from the
cytotoxic effects of 5-Fu, whereas little protection (<5%) of
LPP-CFC was observed in the absence of MPIF-1 or in the presence of
an unrelated protein. HPP-CFC under the same culture conditions
were not protected by MPIF-1, demonstrating the specificity of the
MPIF-1 effect. A similar experiment was also performed with human
CD34.sup.+ cells, where protection from the 5-Fu-induced
cytotoxicity was determined by measuring proliferative capacity of
the survival of cells in response to multiple cytokines. Data
presented in FIG. 18 demonstrates that progenitors capable of
responding to multiple cytokines were protected by about 40% in the
presence of MPIF-1, with little protection observed either in the
absence of any added chemokine or in the presence of MIP-1.alpha..
Together, these data demonstrate that MPIF-1, isolated by the
method of the present invention, is biologically active.
[0226] Effect of MPIF-1 Treatment in Normal Mice
[0227] To determine the effect of administering MPIF-1 purified
according to the present invention on hematopoietic parameters in
vivo, four mice were injected IP twice a day at 8 hours intervals
for two days with either MPIF-1 (0.5 mg/Kg/injection) or saline.
Approximately 12 hours after the last injection, peripheral blood
and bone marrow cells were obtained from each of the animals and
then assayed for the presence of HPP-CFC and LPP-CFC. Table 7 shows
pooled data obtained from four animals in each group and are
expressed as Mean.+-.S.D. Frequency of progenitors in the
peripheral blood was not affected in response to MPIF-1 injections.
However, the frequency of LPP-CFC in the bone marrow of the MPIF-1
injected animals was significantly reduced compared to that found
in animals injected with saline (Table 7).
[0228] Effect of MPIF-1 Pre-Treatment of Mice on the Recovery from
5-Fu-Induced Neutropenia
[0229] The results shown above suggested that the myelotoxicity
elicited by cytotoxic drugs such as 5-Fu, a severe side effect
frequently observed in cancer patients undergoing chemotherapy,
might be ameliorated if the critical cell types within the bone
marrow could be protected during the period of action of the
chemotherapeutic drugs by MPIF-1 purified according to the present
invention. To explore this possibility, a group of mice (Group 4)
were injected (I.P.) daily for three days, at 24 hour intervals,
with 1.0 mg/Kg MPIF-1. On the third day these mice were also
injected (I.P.) with 5-Fu at 150 mg/Kg. Animals injected with
either saline (Group 1), MPEF-1 alone (Group 2), or 5-Fu alone
(Group 3) served as controls. Four animals from each of the groups
were sacrificed at 3, 6, & 10 days post 5-Fu administration to
determine White Blood Cell (WBC) counts in the peripheral blood. As
shown in Table 8, injection of MPIF-1 alone had little effect on
the WBC counts. As expected, 5-Fu treatment resulted in a dramatic
reduction in the circulating WBC counts on day 6 post 5-Fu
administration compared to animals injected with saline.
Significantly, animals treated with MPIF-1 prior to 5-Fu
administration exhibited about two fold higher WBC counts in the
blood compared to animals treated with 5-Fu alone. Thus, treatment
of mice with MPIF-1 prior to 5-Fu apparently resulted in an
accelerated recovery from neutropenia.
[0230] Effect of MPIF-1 Pre-Treatment on the Bone Marrow
Recovery
[0231] Hematopoietic stem and multipotential progenitor cells in
the bone marrow are responsible for restoring all the hematopoietic
lineages following chemotherapy. In normal individuals, these cells
divide less frequently and are therefore spared from a single dose
of a chemotherapeutic drug. However, these cells are killed if a
second dose of the drug is administered within three days after the
first dose is administered as the critical cell types in the bone
marrow are rapidly dividing during this period. To demonstrate that
MPIF-1 purified according to the present invention is able to
protect these cell types in the bone marrow, the following
experiment was performed as illustrated below.
[0232] Three groups of mice (9 animals per group) were treated as
follows: Group 1 was injected with saline on days 1, 2, and 3;
Group 2 was injected with 5-Fu on days 0 and 3; and Group 3 was
injected with 5-Fu on days 0 and 3 and MPIF-1 on days 1, 2, and 3.
Four animals from each group were bled and their bone marrow
harvested 3 and 7 days post second 5-Fu administration to determine
WBC counts, bone marrow cellularity, and HPP-CFC and LPP-CFC
frequencies in the bone marrow using a clonogenic assay. As shown
in Table 9, injection of two doses of 5-Fu to mice resulted in a
dramatic reduction in the peripheral blood WBC counts, total bone
marrow cellularity, and HPP-CFC and LPP-CFC frequencies compared to
that in the saline-injected animals at 3 days post second 5-Fu
administration. These parameters were not significantly affected at
3 days post second 5-Fu in animals pre-treated with MPIF-1 (Table
9). The most pronounced effect of MPIF-1 pre-treatment, especially
with respect to the progenitor frequencies, was observed at 7 days
post second 5-Fu administration; HPP-CFC and LPP-CFC frequencies in
5-Fu treated mice were still at <50% of the control levels,
whereas these progenitor frequencies in mice pre-treated with
MPIF-1 had been restored to those found in the saline injected
control animals (Table 9). A consistent finding has been the
apparent recovery of HPP-CFC from the 5-Fu-induced cytotoxicity in
the MPIF-1 pre-treated animals. This is a surprising result since
MPIF-1 had no impact on colony formation by HPP-CFC in vitro.
[0233] Induction of Calcium Mobilization by MPIF-1 in Human
Monocytes
[0234] Many cell-surface receptors transmit their signals via
G-proteins activate phosphoinositidase C, which catalyzes the
hydrolysis of phosphatidylinositol 4,5-bis-phosphate to the second
messengers, inositol 1,4,5-triphosphate (IP.sub.3) and
diacylglycerol. IP.sub.3 interacts with a specific receptor
population of ligand-gated channels to mobilize non-mitochondrial
intracellular Ca.sup.++ stores (calciosomes and endoplasmic
reticulum). The Indo-1 assay utilizes the highly fluorescent
Ca.sup.++-indicator dye Indo-1 to detect the release and uptake of
native Ca.sup.++ from the intracellular Ca.sup.++ stores. Indo-1
excitation is at 330 nm while detection of bound dye is measured at
405 nm and free dye is measured at 485 nm.
[0235] Chemokines play fundamental roles in the physiology of acute
and chronic inflammatory processes by attracting and stimulating
leukocytes. The actions of chemokines are mediated by G
protein-coupled receptors. Transient increases in cytosolic free
calcium are early events in signal transduction during leukocyte
activation with chemokines. Therefore, the Indo-1 calcium
mobilization assay can be used to assess the effects of MPIF-1
purified according to the present invention on leukocytes.
[0236] Methods
[0237] Cells. A human monocytic-like cell line THP-1 was obtained
from the American Type Culture Collection (ATCC TIB 202) and
maintained in RPMI-1640 medium supplemented with 10% FCS. Human
peripheral blood monocytes were purified from normal blood by
elutriation. The monocyte preparations were 60-80% pure as assessed
by monocyte-specific antibody staining.
[0238] Analysis of intracellular calcium mobilization. Human
monocytes or THP-1 cells were loaded with Indo-1 by incubation for
30 minutes at 37.degree. C. with 2.5 .mu.M
Indo-1/acetoxymethylester per 10.sup.7 cells in HBSS containing 1
mM CaCl.sub.2, 2 mM MgSO.sub.4, 5 mM glucose and 10 mM HEPES. The
cells were washed with HBSS and resuspended in the same buffer at
2-5.times.10.sup.5 cells/ml and stimulated with the indicated
chemokines at 37.degree. C. The fluorescence signals relating to
changes in intracellular calcium ([Ca.sup.++].sub.i) were measured
using a Hitachi F-2000 fluorescence spectrophotometer by monitoring
Indo-1 excitation at 330 nm with detection of fluorescence emitted
at 405 and 485 nm.
[0239] MPIF-1 preparations. MPIF-1 protein preparations expressed
in insect cells transfected with recombinant baculovirus
(HG00300-B5, HG00300-B7 and HG14800-B1) were purified according to
the invention. Proteins released into culture supernatants were
purified and characterized with respect to N-terminal sequence.
Preparation HG00300-B5 was found to contain RVTKDAET (SEQ ID NO: 5)
as the N-terminal amino acid sequence. In contrast, preparation
HG00300-B7 was heterogenous with respect to N-terminal sequence
(four N-truncated species). Preparation HG14800-B1 was from an
alternate spliced cDNA construct. Preparation HG00302 was full
length MPIF-1 with an additional methionine at the N-terminus and
HG00304 was MPIF-1d23, a mutant lacking 23 N-terminal amino acids,
were expressed in E.coli encoded by the cDNA constructs and
purified according to the process of the present invention.
[0240] Results
[0241] MPIF-1 purified according to the invention (HG00300-B5)
induced a rapid calcium mobilization in THP-1 cells at a
concentration of 100 ng/ml. Subsequently, we compared the effect of
MIP-1.alpha. and MPIF-1 on calcium mobilization in THP-1 cells
(FIG. 20) and monocytes (FIG. 21). The minimum concentrations of
MIP-1.alpha. which stimulate calcium mobilization are 10 ng/ml and
1 ng/ml in the monocytes and THP-1 cells, respectively. MPIF-1 was
about 10-fold less potent as compared with MIP-1.alpha.. Prior
incubation of the THP-1 cells with 1000 ng/ml MPIF-1 prevented 50%
of the subsequent response to 100 ng/ml MIP-1.alpha. (FIG. 22A and
FIG. 22B). In contrast, the subsequent response to 1000 ng/ml
MPIF-1 was abolished when the cells were first stimulated with 100
ng/ml MIP-1.alpha.. Similar desensitization results were obtained
with monocytes (FIG. 23A and FIG. 23B). BB100.10 protein was
identical to MIP-1.alpha. except for a difference of one amino acid
at position 26 from Asp to Ala.
[0242] The effects of various MPIF-1 proteins from different cDNA
constructs on monocytes and THP-1 cells are summarized in Table 10
and Table 11, respectively. The order or potency is
HG00300-B7>HG00304>- HG00302. Thus, the N-terminal truncated
MPIF-1 proteins seem to be more active than the full length MPIF-1
protein.
8TABLE 10 EFFECTS OF VARIOUS MPIF-1 PROTEINS ON CALCIUM
MOBILIZATION IN THP-1 CELLS Change in Ca.sup.++ nM Date ng/ml
00300-B5 00300-B7 00302-E1 00302-E2 00304-E1 00304-E2 Jul. 19, 1995
100 140 1,000 Aug. 1, 1995 100 150 1,000 Aug. 18, 1995 100 150
1,000 Apr. 29, 1996 100 50 (21%) 240 (100%) 70 (29%) 1,000 190
(79%) 230 (96%) 200 (83%) May 6, 1996 100 170 (106%) 1,000 May 6,
1996 100 170 (113%) 100 (56%) 1,000 200 (133%) Jun. 24, 1996 10 100
130 (72%) 50 (28%) 40 (22%) 100 (56%) 80 (44%) 1,000 170 (95%) 80
(44%) 80 (44%) 130 (72%) 130 (72%) The numbers in parentheses
represent percentage of MIP-1.alpha. (100 ng/ml) response
[0243]
9TABLE 11 EFFECTS OF VARIOUS MPIF-1 PROTEINS ON CALCIUM
MOBILIZATION IN MONOCYTES Change in Ca.sup.++ nM Date ng/ml
00300-B7 00302-E1 00302-E2 00304-E1 00304-E2 BB100.10(E1) CKB8-AS
May 23, 1996 10 170 (155%) 110 (100%) 100 210 (190%) 40 (36%) 200
(182%) 1,000 220 (200%) 100 (91%) 220 (200%) Jun. 10, 1996 100 200
(133%) 160 (107%) Jun. 13, 1996 10 100 (56%) 100 140 (78%) 1,000
160 (89%) Jun. 18, 1996 10 100 200 (100%) 0 70 (35%) 60 (30%) 1,000
80 (40%) 120 (60%) 140 (70%) Jun. 20, 1996 10 90 (53%) 60 (35%) 20
(12%) 100 210 (123%) 140 (82%) 140 (82%) 1,000 250 (147%) 140 (82%)
0 130 (77%) Jun. 25, 1996 10 160 (89%) 80 (45%) 100 200 (111%) 0 0
50 (28%) 50 (28%) 170 (94%) 1,000 230 (128%) 0 60 (33%) 180 (100%)
190 (105%) 270 (150%) The numbers in parentheses represent
percentage of MIP-1.alpha. or CKB10 (100 ng/ml) response
[0244] Effect of MPIF-1 on the Chemotaxis of Peripheral
Leukocytes
[0245] Chemotaxis refers to the migration of a given cell type in
response to a soluble factor. Upon bacterial infection or tissue
insult, a number of chemotactic factors are released from the
vascular endothelial cells as well as from the responding immune
cells. These factors are thought to be involved in the recruitment
of immune cells to the site of injury or infection and thus play
critical roles in host immune response. In addition, these
chemotactic agents most likely play a role in the normal
trafficking of a variety of immune cell types. MPIF-1 purified
according to the present invention was examined for its chemotactic
activity or peripheral blood mononuclear cells (PBMCs), neutrophils
(PMNs), monocytes, and purified T-lymphocytes as described in the
methods section.
[0246] Method
[0247] Primary Cell Isolation: Cell types used for the chemotaxis
assay include peripheral blood mononuclear cells (PBMCs),
T-lymphocytes, peripheral multi-nucleated cells (PMNs or
neutrophils), and monocytes. Peripheral blood mononuclear cells
were purified from donor leukopacks (Red Cross) by centrifugation
on lymphocyte separation medium (LSM; density 1.077 g/ml; Organon
Teknika Corp.) and harvesting the interface band. Neutrophils were
recovered from the pellet following a dextran sedimentation step
either prior to or after ficoll separation. Monocytes were purified
by centrifugal elutriation and T-lymphocytes were purified from the
PBMCs using T-cell enrichment columns (R&D Systems).
[0248] Chemotaxis assay: Cells used for the assay were washed
3.times. with HBSS/0.1% BSA and resuspended at 2.times.10.sup.6/ml
for labeling. Calcein-AM (Molecular Probes) was added to a final
concentration of 1 .mu.M and the cells incubated at 37.degree. C.
for 30 minutes. Following this incubation, the cells were washed
3.times. with HBSS/0.1% BSA. Labeled cells were resuspended at
4-8.times.10.sup.6/ml and 25 .mu.l (1-2.times.10.sup.5 cells) added
to the top of a polycarbonate filter (5-8 .mu.m pore size, PVP
free; NeuroProbe, Inc.) which separates the cell suspension from
the chemotactic agent in the plate below. Cells were allowed to
migrate for 45-90 minutes and then the number of migrated cells
(both attached to the filter as well as in the bottom plate) was
quantitated using a Cytofluor II fluorescence plate reader
(PerSeptive Biosystems).
[0249] Results
[0250] MPIF-1 purified according to the present invention had
chemotactic activity on PBMCs in a dose range between 30-100 ng/ml
with a chemotactic index (C.I.) ranging from 2-4 times the
background values. fMLP served as a positive control in these
assays and typically had a C.I. of 4-6 times background values. A
representative experiment with PBMCs is shown in FIG. 24. MPIF-1
also showed chemotactic activity in assays with PMNs (FIG. 25) but
this activity was weak (C.I.=2) and not always consistent. fMLP
typically had a C.I. of 6-10 on PMNs and IL-8 typically showed a
C.I. of 8-12 in this assay.
[0251] The PBMC population was further fractionated into
T-lymphocytes and monocytes and the purified populations used for
chemotaxis assays. As shown in FIG. 26, MPIF-1 showed no
chemotactic activity on purified monocytes. In contrast, MPIF-1 had
chemotactic activity on the purified T-lymphocyte population (FIG.
27). This activity was similar to that seen with PBMCs but was
evident at lower MPIF-1 concentrations (3-30 ng/ml) and with a
lower C.I. (2-3 versus 2-4).
[0252] Conclusions
[0253] MPIF-1 purified according to the present invention retains
its biological activity as a potent and specific inhibitor of a
myeloid progenitor that gives rise to colonies composed of both
granulocyte and monocyte lineage cells in vitro. This inhibition
appears to be reversible in nature, as MPIF-1 purified according to
the present invention was able to partially protect these
progenitors from 5-Fu-induced cytotoxicity in vitro as well as in
vivo. MPIF-1 was 10-fold less potent as compared with the same dose
of MIP-1.alpha. inducing calcium mobilization in the monocytes and
THP-1 cells. MPIF-1 and MIP-1.alpha. showed bi-directional cross
desensitization indicating that they share a common receptor.
MPIF-1 also shows chemotactic activity on an unfractionated
population of PBMCs as well as a more purified population of
T-lymphocytes. MPIF-1 has weak chemotactic activity on PMNs and
shows no chemotactic activity on purified monocytes.
[0254] MPIF-1 Mutants
[0255] MPIF-1 N-terminal deletion mutants were also expressed and
purified according to the present invention from baculovirus,
E.coli and CHO expression systems. These mutants were also tested
for biological activity. Particularly, MPIF-1d23 (HG00304), a
mutant having 23 N-terminal amino acids deleted, was purified from
E. coli. This protein was found to be more potent in calcium
mobilization assays with THP-1 cells and monocytes. In addition,
this protein was 10-fold more potent in LPP-CFC inhibition and
protection of myeloid progenitors from 5-Fu-induced cytotoxicity.
MPIF-1d23 was also found to be more active in animal
experiments.
[0256] It will be clear that the invention may be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0257] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the appended claims.
[0258] The disclosures of U.S. application Ser. Nos. 08/722,719,
now U.S. Pat. No. 6,001,606, and 08/722,723, now abandoned, both
filed on Sep. 30, 1996, are incorporated by reference.
Sequence CWU 1
1
5 1 5 PRT Homo sapiens 1 Met Gln Gly Glu Asn 1 5 2 5 PRT Homo
sapiens 2 Thr Asp Gln Leu Ser 1 5 3 216 PRT Homo sapiens 3 Met Gly
Ala Ala Arg Leu Leu Pro Asn Leu Thr Leu Cys Leu Gln Leu 1 5 10 15
Leu Ile Leu Cys Cys Gln Thr Gln Gly Glu Asn His Pro Ser Pro Asn 20
25 30 Phe Asn Gln Tyr Val Arg Asp Gln Gly Ala Met Thr Asp Gln Leu
Ser 35 40 45 Arg Arg Gln Ile Arg Glu Tyr Gln Leu Tyr Ser Arg Thr
Ser Gly Lys 50 55 60 His Val Gln Val Pro Gly Arg Arg Ile Ser Ala
Thr Ala Glu Asp Gly 65 70 75 80 Asn Lys Phe Ala Lys Leu Ile Val Glu
Thr Asp Thr Phe Gly Ser Arg 85 90 95 Val Arg Ile Lys Gly Ala Glu
Ser Glu Lys Tyr Ile Cys Met Asn Lys 100 105 110 Arg Gly Lys Leu Ile
Gly Lys Pro Ser Gly Lys Ser Lys Asp Cys Val 115 120 125 Phe Thr Glu
Ile Val Leu Glu Asn Asn Tyr Thr Ala Phe Gln Asn Ala 130 135 140 Arg
His Glu Gly Trp Phe Met Val Phe Thr Arg Gln Gly Arg Pro Arg 145 150
155 160 Gln Ala Ser Arg Ser Arg Gln Asn Gln Arg Glu Ala His Phe Ile
Lys 165 170 175 Arg Leu Tyr Gln Gly Gln Leu Pro Phe Pro Asn His Ala
Glu Lys Gln 180 185 190 Lys Gln Phe Glu Phe Val Gly Ser Ala Pro Thr
Arg Arg Thr Lys Arg 195 200 205 Thr Arg Arg Pro Gln Leu Pro Thr 210
215 4 7 PRT Homo sapiens 4 Arg Val Thr Lys Asp Ala Glu 1 5 5 8 PRT
Homo sapiens 5 Arg Val Thr Lys Asp Ala Glu Thr 1 5
* * * * *