U.S. patent application number 10/287118 was filed with the patent office on 2003-07-24 for purification of human troponin i.
Invention is credited to Conn, Gregory, Reardon, Brian, Zeng, Xianfang, Zhang, Chenming.
Application Number | 20030138907 10/287118 |
Document ID | / |
Family ID | 22809562 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138907 |
Kind Code |
A1 |
Conn, Gregory ; et
al. |
July 24, 2003 |
Purification of human troponin I
Abstract
The invention is directed to methods for purifying Troponin I,
particularly recombinant Troponin I produced in a bacterial
expression system. Recombinant Troponin I can be advantageously
purified after reversibly protecting the free sulfhydryl groups,
e.g., by forming sulfates. In a specific example, Tropnin I reacted
with sodium tetrafhionate yields sulfitolyzed Tropnin I, which was
purified by chromatography on an anion exchanger, followed by
hydrophobic interaction chromatography. Facile deprotection of the
sulfhydryl groups yields a highly purified product ready for
refolding.
Inventors: |
Conn, Gregory; (Cary,
NC) ; Reardon, Brian; (Seattle, WA) ; Zeng,
Xianfang; (Northborough, MA) ; Zhang, Chenming;
(Blacksburg, VA) |
Correspondence
Address: |
INTERVET INC
405 STATE STREET
PO BOX 318
MILLSBORO
DE
19966
US
|
Family ID: |
22809562 |
Appl. No.: |
10/287118 |
Filed: |
November 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10287118 |
Nov 4, 2002 |
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09998619 |
Nov 30, 2001 |
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09998619 |
Nov 30, 2001 |
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09903398 |
Jul 10, 2001 |
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60217069 |
Jul 10, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/252.33; 435/320.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/1709 20130101;
C07K 14/4716 20130101 |
Class at
Publication: |
435/69.1 ;
435/252.33; 435/320.1; 530/350; 536/23.5 |
International
Class: |
C12P 021/02; C12N
001/21; C07K 014/47; C07H 021/04 |
Claims
What is claimed is:
1. A method of preparing Troponin I, which method comprises
protecting sulfhydryl groups of Troponin I under reducing
conditions.
2. The method according to claim 1, wherein the free sulfhydryl
groups are protected by sulfitolyzation.
3. The method according to claim 2, wherein sulfitolyzation
comprises reacting reduced recombinant Troponin I with sodium
tetrathionate.
4. The method according to claim 1, wherein the recombinant
Troponin I is expressed in a bacterial expression system.
5. The method according to claim 4, wherein the bacterial
expression system is an E. coli expression system.
6. The method according to claim 1, which further comprises
purifying the sulfhydryl-protected recombinant Troponin I.
7. The method according to claim 6, wherein the Troponin I is
purified by chromatography.
8. The method according to claim 6, which comprises purifying the
Troponin I under non-reducing conditions.
9. The method according to claim 6, which further comprises
deprotecting the sulfhydryl groups.
10. Troponin I comprising sulfhydryl protecting groups.
11. The Troponin I of claim 10, which is denatured.
12. The Troponin I of claim 10, wherein the sulfhydryl protecting
groups are sulfates.
13. A method of purifying Troponin I, which method comprises
subjecting Troponin I comprising sulfhydryl protecting groups to
chromatography.
14. The method according to claim 13, wherein the sulfhydryl groups
are protected by sulfitolyzation.
15. The method according to claim 14, wherein sulfitolyzation
comprises reacting reduced, denatured recombinant Troponin I with
sodium tetrathionate.
16. The method according to claim 13, which comprises subjecting
the Troponin I to chromatography under non-reducing conditions.
17. The method according to claim 13, wherein the Troponin I is
expressed in a bacterial expression system.
18. The method according to claim 17, wherein the bacterial
expression system is an E coli expression system.
19. The method according to claim 13, wherein a chromatographic
support is an anion exchange column.
20. The method according to claim 19, which further comprises
chromatography on a hydrophobic interaction chromatographic
support.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to methods for purifying
human Troponin I.
BACKGROUND OF THE INVENTION
[0002] Troponin I (TnI) is a component of a heterotrimeric complex,
along with troponin C (TnC) and troponin T (TnT), involved in
regulation of vertebrate striated muscle contraction (Zot and
Potter, Annu. Rev. Biophys. Biophys. Chem. 1987, 16:535-559; Farah
and Reinah, FASEB J. 1995, 9:755-767). Muscle contraction is
triggered by the binding of Ca.sup.++ ions to TnC. TnT binds to
tropomyosin anchoring the Tn to the muscle filament. TnI is the
inhibitory subunit of the troponin complex, binding to
actin-tropomyosin complexes and preventing the interaction of actin
and myosin. TnI is present in muscle tissue in multiple isoforms
expressed from a multi-gene family (Wu el al., DNA Seq. J. DNA Seq.
Mapp. 1993, 4:113-121).
[0003] Recent investigations have demonstrated a second important
biological function for TnI, the ability of the molecule to inhibit
both in vitro endothelial cell survival/proliferation and inhibit
in vivo angiogenesis (the growth and development of blood vessels)
(Moses et al., Proc. Natl. Acad. Sci. USA 1999, 96:2645-50). TnI
that inhibited endothelial cell development was isolated initially
from cartilagenous tissue, and subsequent studies demonstrated
anti-angiogenic activity with recombinant forms of TnI expressed in
E. coli.
[0004] The TnI subunit is a single polypeptide with a molecular
weight of 21,338. The molecule contains three cysteine residues, at
positions 45, 65, and 134 (Wilkinson and Grand, Biochem J. 1975,
149:493-496). Purification processes previously developed to
isolate native TnI from tissue sources or recombinant TnI require
maintenance of a reducing environment throughout the purification
and storage of TnI, ordinarily by the addition of dithiothreitol
(DTT) to protein preparations (Potter, Methods Enzymol., 1982,
85:241-263; Jha el al., Protein Exp. Purif., 1994, 5:604-613;
Al-Hillawi el al., Eur. J. Biochem., 1994, 225:1195-1201). The
added DTT maintains the TnI cysteine sidechain sulfhydryls in their
reduced state, preventing the formation of intra- or intermolecular
disulfide bond crosslinks. Disulfide bonds between TnI cysteine
sidechains are not believed to be present in the active
conformation of the protein involved in regulation of muscle
contraction (Kluwe et al., FEBS Lett., 1993, 323:83-88), and
reductant was utilized in the isolation of active anti-angiogenic
forms of TnI.
[0005] The necessity of maintaining a reducing environment in
processing and storage during large scale purification of TnI for
commercial use poses numerous technical difficulties and increases
production cost. Thus, there is a need in the art to purify and
refold Tropinin I efficiently and affordably.
SUMMARY OF THE INVENTION
[0006] The present invention provides a method of preparing
Troponin I. This method comprises protecting sulfhydryl groups of
reduced Troponin I, particularly recombinant TnI. In a preferred
embodiment of the invention, the free sulfhydryl groups are
protected by sulfitolyzation of Troponin I expressed in a bacterial
expression system. Protection of the sulfhydryl groups during
Troponin I preparation obviates the costly need for maintaining
non-reducing conditions throughout protein preparation,
purification, and storage.
[0007] In a related aspect, the present invention encompasses
sulfhydryl-protected Troponin I itself, and in a preferred
embodiment, the Troponin I is denatured and the sulfhydryl groups
protected by sulfates.
[0008] The present invention also provides a method of purifying
Troponin I, which method comprises subjecting recombinant Troponin
I comprising sulfhydryl protecting groups to chromatography. In a
preferred embodiment of the invention, the sulthydryl groups are
protected by sulfitolyzation (e.g., via reaction with sodium
tetrathionate). In one aspect of the invention, the Troponin I is
subjected to chromatography under non-reducing conditions. In a
preferred embodiment of the invention the Troponin I to be purified
is expressed in a bacterial expression system such as E coli. In
another preferred embodiment, the chromatographic support is an
anion exchange column, optionally followed by hydrophobic
interaction chromatography.
[0009] These and other aspects of the invention are more fully
examined in the accompanying Drawings, Detailed Description, and
Example.
DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A and 1B. The chemical structure of modified
cysteine. A. Conversion of cysteine to S-sulfocysteine by reaction
with sodium tetrathionate and reversal by exogenous thiols. B. The
cleavage of disulfide bonds by sodium sulfite to form the S-sulfo
derivative.
[0011] FIG. 2. Preparation and washing of TnI-containing inclusion
bodies.
[0012] FIG. 3. Summary of rTroponin-I preparation.
[0013] FIG. 4. Q-Sepharose FF chromatography Troponin I. Buffer A:
6 M urea, 25 mM Tris-HCl, pH 7.5, 100 mM; Buffer B: 6M urea, 25 mM
Tris-HCl, pH 7.5, 2M NaCl; Gradient: Step; 0% B for the
flow-through and 100% B for the strip; and Flow rate: 150
ml/min.
[0014] FIG. 5. 300 ml Q-sepharose FF chromatography. Buffer A: 6M
urea, 25 mM Tris-HCl, pH 7.5, 100 mM; Buffer B: 6M urea, 25 mM
Tris-HCl, pH 7.5, 2M NaCl; Gradient: Step; 4% B for elution and 50%
B for strip; and Flow rate: 20 ml/min.
[0015] FIG. 6. SDS-PAGE analysis troponin lot after anion exchange
steps no. 1 and no. 2 in 16% tris-glycine gel, under non-reducing
conditions. A-H refer to lanes in the SDS-PAGE gel. A. Sulfitolyzed
troponin Lot 3L4 standard; B. solubilized inclusion bodies; C.
sulfitolyzed inclusion bodies (AEX No. 1 load); D. anion exchange
no. 1 flowthrough; E. anion exchange no. 1 salt eulate; F. anion
exchange no. 2 load; G. anion exchange no. 2 flowthrough; and, H.
anion exchange no. 2 100 mM NaCl eluate.
[0016] FIG. 7. Toyopearl 650M (phenyl) HIC chromatograph. Buffer A:
6M urea, 25 mM Tris-HCl, pH 7.5, 1M (NH.sub.4).sub.2SO.sub.4;
Buffer B: 6M urea, 25 mM Tris-HCl, pH 7.5; Gradient: Step; 100% B
for the flow-through and 0% B for strip; and Flow rate: 10
ml/min.
[0017] FIG. 8. SDS-PAGE analysis of troponin lot after hydrophobic
interaction chromatography is a 16% tris-glycine gel, under
non-reducing conditions. A-F refers to lanes in the SDS-PAGE gel.
A. Sulfitolyzed troponin Lot 3L4 standard; B. AEX step no. 2,
troponin eulate pool; C. HIC load (w/1M ammonium sulfate); D. HIC
flowthrough (troponin product); E. HIC low salt eulate (column
strip); F. lot 3L5 sulfitoylzed troponin product.
[0018] FIG. 9. Quantitation of rTnI on Zorbax C3.
[0019] FIG. 10. Troponin I LysC mapping.
[0020] FIG. 11. SDS-PAGE analysis of sulfitolyzed troponin
reduction with dithiothreitol for 45 mins. at ambient temperature.
One mg/ml TnI in 6M urea, 25 mM tris, 0.15M NaCl pH 7.5, run on a
16% tris-glycine gel.
DETAILED DESCRIPTION
[0021] Troponin I from human cartilage has recently been reported
to possess antiangiogenic activity. In order to produce protein to
exploit the antiangiogenic properties of recombinant troponin I, we
overexpressed a human skeletal troponin I cDNA in E. coli.
Expression levels ranged from 2-10 mg/gram of wet cell paste. The
recombinant troponin I was isolated from the lysed cells in
inclusion bodies, which were solubilized and modified by
sulfitolyzation of cysteine residues to improve protein processing.
The sulfitolyzed protein was purified from the inclusion bodies by
sequential anion exchange and hydrophobic interaction
chromatography. Cysteine protecting groups could be removed by
reduction prior to final protein formulation. Overall yield of
troponin from the multi-step purification was greater than 50% at
purity levels of greater than 95%. The purified recombinant human
troponin I is structurally characterized by LC/MS, peptide mapping,
capillary electrophoresis, SEC with laser light scattering
detection, and SDS-PAGE.
[0022] The present invention provides a method to purify and refold
recombinant TnI which obviates the need for reducing agents by
utilizing sulfhydryl protecting groups on the reduced protein, in
particular by oxidative sulfitolysis (Chan, Biochemistry, 1968,
7(12):4247-4253) (FIG. 1). This embodiment involves an initial
modification of the cysteines in troponin to yield stable
S-sulfonated sidechains, which are maintained on the protein during
processing and storage. The sulfate protecting groups can be
removed from the TnI cysteines by treatment with a reductant to
regenerate the free cysteine sulfhydryls.
[0023] The term "purified" as used herein refers to material that
has been isolated under conditions that reduce or eliminate
unrelated materials, i.e., contaminants. For example, a purified
protein is preferably substantially free of other proteins or
nucleic acids with which it is associated in a cell. As used
herein, the term "substantially free" is used operationally, in the
context of analytical testing of the material. Preferably, purified
material substantially free of contaminants is at least 50% pure;
more preferably, at least 90% pure, and more preferably still at
least 99% pure. Purity can be evaluated by chromatography, gel
electrophoresis, immunoassay, composition analysis, biological
assay, and other methods known in the art.
[0024] Recombinant TnI can be expressed in bacterial systems in a
soluble form and an insoluble form, in inclusion bodies. Recovery
of TnI from inclusion bodies requires treatment with solubilizing
protein denaturants like urea. In addition, TnI, whose theoretical
pI is 8.8, has limited solubility at pH values above 4 in the
absence of chaotropic agents, although TnI is soluble at levels of
10-20 mg/ml at low pH (less than 3). High levels (1-6M) of the
protein denaturant urea are therefore ordinarily maintained during
protein purification of TnI to ensure high solubility and good
protein recovery.
[0025] The term "refolding" means changes in the three-dimensional
conformation of the protein which restore the protein's biological
activity, including its antiangiogenic properties.
[0026] Protein refolding of TnI into a bioactive conformation in a
buffer and at a pH suitable for use in human patients requires
removal of solubilizing denaturant. Circular dichroism and
fluorescence studies of TnI protein folding have shown a non-linear
relationship relative to chaotrope concentration in solution, with
little or no apparent secondary structure at high urea
concentration, and putative intermediate folded state(s) occurring
between 2-4M urea (Morjana and Tal, Biotechnol. Aappl. Biochem.,
1998, 28:7-17). This protein refolding process can be accomplished
by dilution of TnI in a denaturant solution to an appropriate
concentration, generally below 10 nanomolar, followed by removal of
solubilizing chaotrope by dialysis, diafiltration, or gel
filtration. Proper refolding requires appropriately timed transit
through the chaotrope concentrations of 2-4M urea, where protein
folding intermediates are formed, followed by a final protein
concentration step.
[0027] As used herein, the term "recombinant TnI" (or troponin I)
refers to TnI prepared by a biological fermentation process. TnI is
a polypeptide of about 21 kD containing three cysteine residues,
although the present invention encompasses modified forms of TnI
lacking one or two cysteine residues. In a specific embodiment, TnI
refers to the protein described by Wilkinson and Grand (Biochem. J.
1975, 149:493-496). In a preferred embodiment, TnI of the invention
lacks any disulfide bonds.
Recombinant Expression
[0028] As noted above, TnI can be prepared, albeit with certain
difficulties, by fermentation of genetically modified cells.
Preferably the cells are bacterial cells, which, lacking eukaryotic
translational and post-translational machinery, produce improperly
folded TnI that must be refolded. However, any expression system
can be used to produce recombinant TnI, particularly systems that
require denaturation and refolding. Furthermore, this system
advantageously provides an effective technique for purifying
Troponin I from any source, including natural TnI and properly
folded recombinant TnI under normal purification conditions (i. e.,
under non-reducing conditions).
[0029] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA
Cloning. A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic
Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)];
Transcription And Translation [B. D. Hames & S. J. Higgins,
eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];
Immobilized Cells And Enzymes[ IRL Press, (1986)]; B. Perbal, A
Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley &
Sons, Inc. (1994).
[0030] The terms "express" and "expression" mean allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing a protein by activating the
cellular functions involved in transcription and translation of a
corresponding gene or DNA sequence. A DNA sequence is expressed in
or by a cell to form an "expression product" such as a protein. The
expression product itself , e.g. the resulting protein, may also be
said to be "expressed" by the cell.
[0031] The term "expression system" means a host cell and
compatible vector under suitable conditions, e.g. for the
expression of a protein coded for by foreign DNA carried by the
vector and introduced to the host cell. Common expression systems
include E. coli host cells and plasmid vectors, insect host cells
and Baculovirus vectors, and mammalian host cells and vectors. In a
specific embodiment, the protein of interest is expressed in E.
coli bacterial cells.
[0032] The term "host cell" means any cell of any organism that is
selected, modified, transformed, grown, or used or manipulated in
any way, for the production of a substance by the cell, for example
the expression by the cell of a gene, a DNA or RNA sequence, a
protein or an enzyme. Host cells can further be used for screening
or other assays. Host cells can be cultured cells in vitro or one
or more cells in a plant, e.g., a transgenic plant or a transiently
transfected plant. Host cells of the invention include, though they
are not limited to, bacterial cells (e.g., E. coli, Synechocystis
sp., Z. mobilis, Agrobacterium tumefaciens, and Rhodobacter); yeast
cells (e.g., S. cerevisiae, Candida utilis, Phaffia rhodozyma);
fungi (e.g., Phycomyces blakesleeanus); algae (e.g., H. pluvalis);
and plants (e.g., Arabidopsis thaliana).
Sulfhydryl Protecting Groups
[0033] As noted above, the sensitivity of recombinant troponin to
oxidation requires maintenance of reducing conditions during
purification of the protein. This results in significant drawbacks
in terms of expense and difficulty.
[0034] The present invention addresses these deficiencies of the
prior methods by providing sulfhydryl protected recombinant
troponin I.
[0035] The term "sulfhydryl protecting group" or "cysteine
protecting group" means a reversibly bound chemical group which
prevents formation of intra- and intermolecular disulfide bonds,
but does not interfere with the process of protein purification. In
a preferred embodiment, the "sulfhydryl protecting group" consists
of sulfate groups bound through sulfitolyzation with sodium
tetrathionate. Numerous other reversible derivatizing reagents for
cysteine sulfhydryls have been developed including disulfide
compounds such as pyridyl disulfide, and the
alkylalkanethiosulfonates. The sulfyhdryl modification by these
reagents is often facile, although their steric properties may
interfere with protein activity or bioprocessing. Other
sulfhydryl-reactive chemistries with potential utility in
simplifying troponin I purification, recovery and storage include
cyanylation and aminoethylation, reaction with compounds containing
the maleimide functional group such as N-ethyl maleimide, vinyl
sulfones, and alkyl halides such as iodoacetic acid and amide.
However, these sulfhydryl modifying groups have the disadvantage of
poor reaction reversibility, making regeneration of the free
sulfhydryl sidechains of the protein cysteines more difficult.
[0036] Preferably, to ensure complete protection of Troponin I's
sulfhydryl groups and effective chromatographic purification, the
Troponin I is denatured prior to reaction with the protecting
groups under reducing conditions.
Chromatographic Methods
[0037] Anion exchange chromatography, hydrophobic interaction
chromatography and preferably a combination thereof can be used to
purify sulfhydryl protected TnI. Other suitable chromatographic
techniques include cation exchange chromatography, gel permeation
chromatography, reverse phase chromatography, metal chelation
chromatography, etc. These chromatographic techniques can be
employed in various formats, including high performance,
preparative column, bulk suspension, and the like. In addition, the
present invention is amenable to other standard laboratory or
industrial separation techniques.
[0038] In a specific embodiment, sulfhydryl-protected recombinant
TnI is purified by successive chromatographies on an ion exchange
column, particularly an anion exchanger, followed by hydrophobic
interaction chromatography.
[0039] Sulfhydryl-protected TnI can be stored in the protected
state, either before or after chromatography. Storage of the
sulfhydryl-protected TnI obviates the need for maintaining reducing
conditions, and avoids formation of intrachain or interchain
disulfide crosslinks.
[0040] The sulfhydryl-protected TnI can be deprotected after
chromatographic or other purification, or storage, as set forth in
the following section.
TnI Deprotection
[0041] Deprotection of the sulfhydryl protected TnI can be achieved
under reducing conditions, e.g., to remove sulfate groups. The
chemistries effective to remove reversible protecting groups are
well-known in the art. Chemistry to remove these reversible
sulfhydryl modifying groups often involves use of reductants such
as mercaptans or dithiothreitol (Hoppe et al., Biochemistry
1989,28:2956; DiBella et al., J. Biol. Chem. 1995,270:163; Kenyon
and Bruice, Methods Enzymol. 1977,47:407; Bruice and Kenyon, J.
Protein Chem. 1982, 1:47; Inoue el al., Biotechnol. Appl. Biochem.
1998, 28:207).
[0042] Exemplary TnI deprotection: Desulfitolyzation. Troponin may
be stored or used in the sulfitolyzed form; if desulfitolyzed
troponin is required the sulfhydryl group modifications may be
removed by treatment of the protein with reductant such as
mercaptans or dithiothreitol (DTT). At neutral to slightly acidic
pH values desulfitolyzation may be accomplished by treatment of the
protein with millimolar levels of reductant (see FIG. 11) in the
presence or absence of a solublizing chaotrope. In normal practice,
levels 10-100 fold higher are used to ensure complete conversion of
the troponin. At more acidic pH values a reductant like
Tris[2-carboxyethylphosphine] hydrochloride (TCEP) is preferred.
The protein may be buffer exchanged by a method such as
dialysis/diafiltration or gel filtration into an acidic pH buffer
prior to removal of reductant to slow the formation of
intermolecular disulfides.
Protein Refolding/Formulation
[0043] Purified troponin can be refolded, if desired, by first
buffer exchanging the protein by dialysis, diafiltration, gel
filtration or other appropriate technique into a suitable refolding
buffer in the presence of a denaturing chaotrope like 8M urea or 6M
guanidine. The denatured protein may then be refolded by dilution
to a suitable target concentration (<10 nanomolar) in an
appropriate buffer with or without chaotrope (8-0 M urea, 6-0 M
guanidine) at an appropriate temperature, and subjected to a timed
hold. Alternatively, the protein may be subjected to dialysis or
diafiltration through appropriate chaotrope level transitions with
hold times to promote protein refolding in a suitable refolding
buffer. The refolded protein is subsequently concentrated by
ultrafiltration and final formulation buffer exchange, if
necessary, can be accomplished by gel filtration or
diafiltration.
[0044] The highly purified TnI, preferably in a refolded state,
produced according to the invention can be used for any purpose,
including but by no means limited to antibody generation, as a
control or standard immunoassay reagent, or to inhibit angiogenesis
(which can be important in treating various cancers).
EXAMPLE
[0045] The present invention will be better understood by reference
to the following Example, which is provided by way of illustration
and not by way of limitation.
Materials and Methods
[0046] TnI Inclusion Body Preparation. Human skeletal TnI expressed
in E. coli was isolated from lysed cells in inclusion bodies (FIG.
2). To isolate and wash inclusion bodies, approximately 150 grams
of cell paste was dispersed in 1.5 liter of 50 mM sodium acetate, 2
mM EDTA, pH 6. The cell suspension is subjected to two consecutive
passes through a microfluidizer, 10,000 psig at 10-12.degree. C.,
to break open the cells. The resultant lysate was centrifuged at
12,000 G, 4.degree. C. for 30 minutes to pellet insoluble material.
The supernatant was removed and the pelleted material was dispersed
in 1.5 liter 50 mM sodium acetate. 2 mM EDTA, 1% Triton X-100, pH 6
and centrifuged for an additional 30 minutes at 12,000 G. The
pelleted material was recovered, dispersed in 1.5 liter 50 mM
sodium acetate, 2 mM EDTA, 0.5M NaCl pH 6, and centrifuged for 30
minutes at 12,000 G. The pelleted material is again dispersed in
1.5 liter of 50 mM sodium acetate, 2 mM EDTA, pH 6 and centrifuged
for 30 minutes at 12,000 G. The resulting pellet is suspended in
200 ml of 50 mM sodium acetate, 2 mM EDTA, pH 6 and centrifuged at
12,000 G for 30 minutes. The final pelleted inclusion. bodies,
approximately 10 grams, was stored at -70.degree. C. prior to
subsequent processing.
[0047] Inclusion Body Solubilization and Sulfitolyzation. Ten grams
of TnI-containing inclusion bodies were solubilized and protein
sulfhydryls were sulfitolyzed using 200 ml 6M urea, 25 mM Tris, 10
mg/ml sodium sulfite, 5 mg/ml sodium tetrathionate pH 7.5 at
ambient temperature for 6 hours in the dark (FIG. 6). The
solubilized material was filtered over a 0.2 micron membrane prior
to subsequent processing.
[0048] TnI Purifcation. Sulfitolyzed recombinant human TnI was
purified by a five step process (FIG. 3). Solubilized, sulfitolyzed
TnI-containing inclusion bodies (200 ml) were loaded onto a 3 liter
volume Q-Sepharose FF (Pharmacia) column pre-equilibrated in 6M
urea, 25 mM Tris, 0.1M NaCl pH 7.5 at 150 ml/minute (FIG. 4). The
purified TnI was collected in the column flowthrough (approximately
3000 ml, total). The recovered TnI was concentrated and buffer
exchanged by UF/DF using a 0.2 ft.sup.2 Pall Omega cassette.
Initial concentration was to 10.times.volume (300 ml) followed by
diafiltration against 5 liters of 6M urea, 25 mM Tris pH 7.5. This
material was loaded onto a 300 ml volume Q-Sepharose FF column
pre-equilibrated in 6M urea, 25 mM Tris, pH 7.5 at 20 ml/minute.
The bound TnI was eluted from the column by a step wash with 6M
urea, 25 mM Tris, 80 mM NaCl pH 7.5 (FIGS. 5, 6). This eluted
troponin (500 ml) was loaded onto a 60 ml column of Toyopearl 650M
Phenyl HIC resin after addition of ammonium sulfate to a final
concentration of 1 M. The column was pre-equilibrated with 6M urea,
25 mM Tris, 1M ammonium sulfate pH 7.5. The purified troponin was
collected as the unbound flowthrough from this column (FIGS. 7, 8),
concentrated 2.5-fold and buffer exchanged for storage by UF/DF
using a 0.2 ft.sup.2 Pall Omega cartridge against 5 liters of 25 mM
sodium citrate, 150 mM NaCl pH 3. Purified TnI was stored frozen at
-70.degree. C.
[0049] Protein purity was determined by SDS-PAGE (FIG. 8) and
reverse phase chromatography (FIG. 9) and protein identity was
confirmed by peptide mapping with peptide mass and fragmentation
analysis (FIG. 10). Yield determinations for each step in the
process were determined by quantitative reverse phase
chromatography (FIG. 11). Residual DNA levels, measured by DNA
Threshold, were less than or equal to 12 pg DNA/mg protein.
Endotoxin testing of final product by LAL (gel-clot) indicated less
than or equal to 3 EU/mg protein.
[0050] TnI Desulfitolyzation. For removal of sulfate groups from
cysteine sidechains, purified TnI in sodium citrate storage buffer
was first buffer exchanged by dialysis or diafiltration into 8M
urea, 25 mM Tris, 0.15M NaCl pH 7.5. Desulfitolyzation was
accomplished by addition of DTT to a final concentration of 0.1M
and incubation at ambient temperature for 1 hour (FIG. 12). The TnI
solution was subsequently buffer exchanged by dialysis or
diafiltration in the presence of reductant to a pH of 6 (8M urea,
10 mM sodium citrate, 5 mM DTT, 0.15M NaCl, pH 6), then into buffer
minus reductant (8M urea, 10 mM sodium citrate, 0.15M NaCl, pH 6)
to preclude the formation of intermolecular disulfides upon removal
of reductant at high pH.
[0051] Analytical Metliods. SDS-PAGE analysis was performed using
Novex Pre-cast 16% Tris-glycine gels, sample and running buffers
and molecular weight markers. Gels were stained with Novex
colloidal Coomassie blue stain.
[0052] Reverse phase chromatographic analyses were performed on an
HP 1100 liquid chromatograph using a 2.1.times.150 mm Zorbax C3SB
column run at a flowrate of 0.25 ml/minute at 25.degree. C. Buffer
for reverse phase column equilibration was 0.1% TFA in water. Tn I
samples were analyzed by gradient elution from 0.1% TFA/water to
0.1% TFA/acetonitrile at 1%/minute. Column eluates were monitored
by on-line UV detection at 215 nm.
[0053] Peptide maps of Tn I were generated by dilution of stock
samples of TnI to 1 mg/ml with 0.1 M Tris buffer, pH 8, followed by
digestion with a 1:20 enzyme:substrate ratio of endoproteinase LysC
at 37.degree. C. for 6 hours. LysC peptide fragments were resolved
by reverse phase chromatography on a 2.1.times.150 mm Zorbax C18SB
column using a gradient of 0.1% TFA/water to 0.1% TFA/acetonitrile
at 0.25 ml/minute. Eluted peptide fragments of Tn I were identified
by on-line LC/MS detection using a Finnigan LCQ ion trap mass
spectrometer set to perform automated peptide
detection/fragmentation analysis.
[0054] Protein concentration analysis was performed using reverse
phase analysis of TnI on the Zorbax C3SB column, whose linear
detection limits were calibrated for quantitation using a TnI
standard whose concentration was determined by UV 280 nm
measurement and the application of the Beer/Lambert equation
(A=Ebc) with an extinction coefficient of 0.4.
[0055] Residual DNA analysis was performed with DNA Threshold.
Endotoxin contaminant was measured by Limulus Amoebocyte Lysate
(LAL) gel-clot test.
Results
[0056] Samples were removed at each process step for analysis.
Sample quantities were determined by reverse phase chromatography.
The troponin peak absorbance at 215 nm was measured for each sample
in replicate, averaged, and the quantity of protein calculated
using a reverse phase calibration curve based upon a standard
troponin dilution series. The calibrant standard concentration was
determined using the Beer/Lambert equation, the standard absorbance
at 280 nm, and an extinction coefficient of 0.4.
[0057] During final UF/DF processing, product precipitation was
noted. After removing final product, residual troponin precipitate
in the UF/DF cassette was resolubilized by washing with 50 ml of 6M
urea, 10 mM sodium citrate, 0.1 5M NaCl, pH 6. This resolubilized
troponin was buffer exchanged to remove urea and analyzed for
troponin. The product total is the sum of the troponin recovered
during the final UF/DF step and the resolubilized, buffer exchanged
cassette wash.
1 Data Summary Table Lot 3L5 Sulfitolyzed Tropinin Purification
VOLUME CONCENTRATION RECOVERY SAMPLE (ML) MG/ML AMOUNT % Cell
Pellet -- -- 148 grams -- Inclusion Body Final Pellet -- -- 10.48
grams -- Q1 Load, Sulfitolyzed Inclusion 250 2.56 640 mg 100 Bodies
Q1 Flowthrough 3000 0.21 644 mg 100 Q1 UF 300 1.94 582 mg 91 Q1 DF
290 1.99 577 mg 90 Q2 Bound, 80 mM NaCl Eulate 500 0.98 489 mg 76
HIC Load, IM Ammonium Sulfate 500 0.99 499 mg 78 HIC Flowthrough
550 0.87 479 mg 75 Final UF/DF 200 1.60 318 mg 50 Cassette
Wash/Reprocessed 50 0.69 34 mg 5 Precipitate Product Total -- --
352 mg 55
[0058] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0059] It is further to be understood that all values are
approximate, and are provided for description.
[0060] All patents, patent applications, publications, procedures,
and other materials cited herein are hereby incorporated by
reference in their entireties.
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