U.S. patent application number 11/336748 was filed with the patent office on 2006-06-08 for novel method for purification of recombinant proteins.
Invention is credited to Paul S. Nelson, Brian Perry, Thomas H. Smith, Te-Tuan Yang.
Application Number | 20060122378 11/336748 |
Document ID | / |
Family ID | 24806511 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060122378 |
Kind Code |
A1 |
Perry; Brian ; et
al. |
June 8, 2006 |
Novel method for purification of recombinant proteins
Abstract
Purification of poly-amino acid-tagged recombinant proteins has
been improved by the use of a carboxymethylated aspartate ligand
complexed with a third-block transition metal having an oxidation
state of 2.sup.+ and a coordination number of 6. A method for
synthesizing the metal ion-CM-Asp complex is also described.
Further, the metal ion-CM-Asp complex can be used for screening
protein function.
Inventors: |
Perry; Brian; (Fremont,
CA) ; Nelson; Paul S.; (Union City, CA) ;
Yang; Te-Tuan; (Los Altos, CA) ; Smith; Thomas
H.; (San Carles, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVENUE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Family ID: |
24806511 |
Appl. No.: |
11/336748 |
Filed: |
January 19, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09839696 |
Apr 19, 2001 |
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11336748 |
Jan 19, 2006 |
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08912406 |
Aug 18, 1997 |
6242581 |
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09839696 |
Apr 19, 2001 |
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08698747 |
Aug 16, 1996 |
5962641 |
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08912406 |
Aug 18, 1997 |
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Current U.S.
Class: |
530/414 |
Current CPC
Class: |
B01D 15/3804 20130101;
C07K 1/22 20130101; C12N 9/003 20130101; C07K 2319/21 20130101;
B01D 15/3828 20130101; C07K 14/43595 20130101; B01J 45/00 20130101;
G01N 30/02 20130101; G01N 30/02 20130101; B01J 20/3265
20130101 |
Class at
Publication: |
530/414 |
International
Class: |
C07K 1/10 20060101
C07K001/10 |
Claims
1. An immobilized metal ion affinity chromatography purification
method for purification of a recombinant proteins, said method
comprising: (a) providing carboxymethylated aspartate ligand
complexed with a transition metal ion in a 2.sup.+ oxidation state,
having a coordination number of 6; (b) loading a mixture of cell
lysate comprising a recombinant protein having a polyhistidine tail
to bind with said ligand; and (c) eluting said recombinant protein
with a suitable elutant to obtain a purified recombinant
protein.
2. The method, according to claim 1, wherein said transition
metal-complexed carboxymethylated aspartate ligand forms a
carboxymethylated aspartate chelating matrix which comprises said
transition metal and a polymer matrix.
3. The method, according to claim 2, wherein said transition metal
is connected to said polymer matrix by a linking arm and a
functional linking group.
4. The method, according to claim 3, wherein said linking arm is
selected from the group consisting of --CH.sub.2CH(OH)CH.sub.2--,
--CH.sub.2(OH)CH.sub.2--O--CH.sub.2CH(OH)CH.sub.2--,
--(CH.sub.2).sub.4NHCH.sub.2CH(OH)CH.sub.2--, and
--{CH.sub.2).sub.2NHCH.sub.2CH(OH)CH.sub.2--.
5. The method, according to claim 3, wherein said functional
linking group is selected from the group consisting of O, S, and
NH.
6. The method, according to claim 2, wherein said polymer matrix is
agarose.
7. The method, according to claim 2, wherein said carboxymethylated
aspartate chelating matrix has the structure ##STR2## wherein:
R.sub.4--R.sub.5--R.sub.6.dbd.H M=transition metal ion in a 2.sup.+
oxidation state with a coordination number of 6; R.sub.1=a linking
arm connecting the nitrogen atom of CM-Asp with R.sub.2; R.sub.2=a
functional linking group through which CM-Asp linking arm R.sub.1
is connected to R.sub.3; and R.sub.3=a polymer matrix
8. The method, according to claim 2, wherein said carboxymethylated
aspartate chelating matrix has the structure ##STR3## wherein:
R.sub.1--R.sub.2--R.sub.3.dbd.H; M=transition metal ion in a
2.sup.+ oxidation state with a coordination number of 6; R.sub.4=a
linking arm connecting the methylene carbon atom of the
carboxymethyl group of CM-Asp with R.sub.5; R.sub.5=a functional
linking group through which CM-Asp linking arm R.sub.4 is connected
to R.sub.6; and R.sub.6=a polymer matrix.
9. An immobilized metal ion affinity chromatography complex
comprising a carboxymethylated aspartate ligand and a transition
metal complexed thereto, wherein said transition metal ion has a
2.sup.+ oxidation state and a coordination number of 6.
10. The complex, according to claim 9, wherein said complex has the
structure: ##STR4## wherein: R.sub.4--R.sub.5--R.sub.6.dbd.H
M=transition metal ion in a 2.sup.+ oxidation state with a
coordination number of 6; R.sub.1=a linking arm connecting the
nitrogen atom of CM-Asp with R.sub.2; R.sub.2=a functional linking
group through which CM-Asp ticking arm R.sub.1, is connected to
R.sub.3; and R.sub.3=a polymer matrix
11. The method, according to claim 10, wherein said polymer matrix
comprises a polymer matrix suitable for use in affinity or gel
chromatography.
12. The complex, according to claim 10, wherein M=Fe.sup.2+,
Co.sup.2+, Ni.sup.2+, Cu.sup.2+, or Zn.sup.2+;
R.sub.1.dbd.--CH.sub.2CH(OH)CH.sub.2--,--CH.sub.2(OH)CH.sub.2--O--CH.sub.-
2CH(OH)CH.sub.2-- or --(CH.sub.2)NHCH.sub.2CH(OH)CH.sub.2--.
R.sub.2.dbd.O, S, or NH; and R.sub.3=agarose or polystyrene.
13. The complex, according to claim 12, wherein M=Co.sup.2+;
R.sub.1.dbd.CH.sub.2CH(OH)CH.sub.2; R.sub.2.dbd.O; and
R.sub.3=agarose, cross-linked or polystyrene
14-21. (canceled)
22. A method for synthesizing carboxymethylated aspartate chelating
matrices, said method comprising the steps: (a) Michael addition of
the a-amino function of monoprotected .alpha., .omega.-diamino
acids to maleic acid; (b) deprotecting the co-amino functionality;
and (c) attaching the chelator primary amine molecule to a solid
matrix.
23. A method for screening for protein function on a microtiter
plate or filter, said method comprising the steps: (a) immobilizing
a complex of claim 1 to the plate or filter; (b) binding said
immobilized complex to the protein for which the function is being
screened; and (c) performing an assay for protein function on the
bound protein.
Description
CROSS-REFERENCE TO A RELATED APPLICATION
[0001] This application is a continuation-in-part of co-pending
application Ser. No. 08/698,747, filed Aug. 16, 1996.
BACKGROUND OF THE INVENTION
[0002] Immobilized metal ion affinity chromatography (IMAC) was
first introduced by Porath (Porath, J., J. Carlsson, I. Olsson, G.
Belfrage [1975] Nature 258:598-599.) under the term metal chelate
chromatography and has been previously reviewed in several articles
(Porath, J. [1992] Protein Purification and Expression 3:263-281;
and articles cited therein). The IMAC purification process is based
on the employment of a chelating matrix loaded with soft metal ions
such as Cu.sup.2+ and Ni.sup.2+. Electron-donating groups on the
surface of proteins, especially the imidazole side chain of
histidine, can bind to the non-coordinated sites of the loaded
metal. The interaction between the electron donor group with the
metal can be made reversible by lowering the pH or by displacement
with imidazole. Thus, a protein possessing electron-donating groups
such as histidine can be purified by reversible metal
complex/protein interactions.
[0003] Several different metal chelating ligands have been employed
in IMAC to purify proteins. Iminodiacetic acid (IDA) ligand is a
tridentate and thus anchors the metal with only three coordination
sites (Porath, J., B. Olin [1983] Biochemistry 22:1621-1630).
Because of the weak anchoring of the metal, metal leakage has been
known to occur. The tris(carboxymethyl)ethylenediamine (TED) ligand
is pentadentate and forms a very strong metal-chelator complex. The
disadvantage of this is that proteins are bound very weakly since
only one valence is left for protein interaction. Nitrilo triacetic
acid (NTA) is a tetradentate ligand which attempts to balance the
metal anchoring strength with metal-ion protein interaction
properties (Hochuli, E., H. Dobeli, A. Schacher [1987] J.
Chromatography 411:177-184). Other chelating ligands have been
reported and are mentioned. See, e.g., Porath (1992), supra.
However, these ligands also have certain disadvantages, including
decreased bonding capacity, decreased specificity, and increased
metal leakage.
[0004] In 1991, Ford et al. (Ford, C., I. Suominen, C. Glatz [1991]
Protein Expression and Purification 2:95-107) described protein
purification using IMAC technology (Ni-NTA ligand) as applied to
recombinant proteins having tails with histidine residues
(polyhistidine recombinant proteins). This method takes advantage
of the fact that two or more histidine residues can cooperate to
form very strong metal ion complexes. The NTA chelating ligand
immobilized on agarose and loaded with Ni.sup.2+ has been useful in
this method (Hochuli et al., supra; U.S. Pat. No. 5,047,513). It is
available commercially through Qiagen, Inc. (Chatsworth, Calif.).
However, this resin has the disadvantage that the interchanges
between metal ions and poly-histidine recombinant proteins are not
optimal. Metal leakage can occur, and background proteins can
sometimes contaminate purification of recombinant proteins.
[0005] A metal chelating gel, i.e., carboxymethylated aspartate
(CM-Asp) agarose complexed with calcium, has been used for
purifying native calcium-binding proteins (Mantovaara, T., H.
Pertoft, J. Porath [1989] Biotechnology and Applied Biochemistry
11:564-570; Mantovaara, T., H. Pertoft, J. Porath [1991]
Biotechnology and Applied Biochemistry 13:315-322; Mantovaara, T.,
H. Pertoft, 3. Porath [1991] Biotechnology and Applied Biochemistry
13:120-126). However, the Ca.sup.2+-CM-Asp complex described by
Mantovaara et al. has among its disadvantages that it does not bind
strongly to histidine-tagged recombinant proteins. Another
disadvantage, in addition to this inferior binding property, is its
non-selectivity for histidine tags.
[0006] By contrast, the subject invention comprises the CM-Asp
chelating ligand complexed to a transition metal in an octahedral
geometry (coordination number of 6). In this unique configuration,
the metal complex can be advantageously suited for purification of
poly-histidine fused recombinant proteins. This is a novel use of
the CM-Asp ligand and is part of the subject of the invention
herein described.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention concerns a novel IMAC purification
method which employs immobilized carboxymethylated aspartate
(CM-Asp) ligands specifically designed for purification of
recombinant proteins fused with poly-histidine tags. The new
purification method is based upon the CM-Asp chelating matrix
having the following structure: ##STR1##
[0008] A general description of the matrix used in the invention
and illustrated above is: [0009] When R.sub.4--R.sub.5--R.sub.6=H:
[0010] M=transition metal ion in a 2+oxidation state with a
coordination number of 6; [0011] R.sub.1=a linking arm connecting
the nitrogen atom of CM-Asp with R.sub.2; [0012] R.sub.2=a
functional linking group through which CM-Asp linking arm R.sub.1
is connected to R.sub.3; [0013] R.sub.3=a polymer matrix, e.g.,
those polymer matrices typically used in affinity or gel
chromatography. [0014] When R.sub.1-R.sub.2-R.sub.3=H: [0015]
R.sub.4=a linking arm connecting the methylene carbon atom of the
carboxymethyl group of CM-Asp with R.sub.5; [0016] R.sub.5=a
functional linking group through which CM-Asp linking arm R.sub.4
is connected to R.sub.6; [0017] R.sub.6-=a polymer matrix, e.g.,
those polymer matrices typically used in affinity or gel
chromatography.
[0018] In a preferred embodiment: [0019] M=Fe.sup.2+, Co.sup.2+,
Ni.sup.2+, Cu.sup.2+, or Zn.sup.2+; [0020]
R.sub.1.dbd.--CH.sub.2CH(OH)CH.sub.2--,
--CH.sub.2(OH)CH.sub.2--O--CH.sub.2CH(OH)CH.sub.2--,
--(CH.sub.2).sub.4NHCH.sub.2CH(OH)CH.sub.2--, and
--(CH.sub.2).sub.2NHCH.sub.2CH(OH)CH.sub.2--, [0021] R.sub.2.dbd.O,
S, or NH; and [0022] R.sub.3=agarose.
[0023] In a particularly preferred embodiment: [0024] M=Co.sup.2+;
[0025] R.sub.1.dbd.CH.sub.2CH(OH)CH.sub.2; [0026] R.sub.2.dbd.O;
and [0027] R.sub.3=agarose, cross-linked.
[0028] Prior to loading the 6.times.His recombinant protein to the
resin, recombinant cells can be lysed and sonicated. The lysate can
then be equilibrated with an aqueous buffer (pH 8) which itself
does not form chelates with the metal. An example of an aqueous
that can be used at this step in the described procedure is 50 mM
sodium phosphate (pH 8.0)/10 mM Tris-HCl (pH 8.0)/100 mM NaCl, or
the like. The equilibration buffer can contain denaturing agents or
detergents, e.g., 10% "TRITON X-100," 6 M ganidinium HCl, or the
like. After binding the prepared 6.times.His recombinant protein on
the metal CM-Asp chelating resin (the "CM-Asp resin complex"), the
protein-bound resin is washed at pH 7.0 or 8.0. The elution of the
protein can be carried out at a constant pH or with a descending pH
gradient. In a preferred embodiment, protein elution can be
achieved at a pH of about 6.0 to about 6.3. Alternatively, the
6.times.His recombinant protein bound to the CM-Asp chelating resin
can be washed with low concentrations (less than 100 mM) of
imidazole at pH 8.0 and then eluted by increasing the imidazole
concentration to 40-100 mM.
[0029] Also included as an aspect of the subject invention is a
scaled-up synthesis of the CM-Asp derivatized agarose chelating
resin. It is an improved version of a previously reported small
scale preparation (Mantovaara, T., H. Pertoft, J. Porath [1991]
Biotechnology and Applied Biochemistry 13:315-322). The improvement
includes particular conditions for oxirane-agarose formation,
temperature controlled conjugation of aspartic acid to the
oxirane-agarose, and high ionic strength washing to remove
extraneously bound metals. These conditions, temperatures, and
ionic concentrations are described in detail herein.
[0030] An additional application of the subject invention includes
screening for protein function on a microtiter plate or filter. The
additional applications for the subject invention also include
protein-protein interaction studies, as well as antibody and
antigen purification. For example, by immobilization of the
Co.sup.2+ moiety onto 96-well plates by CM-Asp, such plates can be
used for quantitation of 6.times.Histidine-tagged protein,
protein-protein interaction studies, diagnostic screening for
diseases, antibody screening, antagonist and agonist screening for
drugs, and reporter gene assays. Co.sup.2+ can also be immobilized
onto a membrane, e.g., a nylon membrane, by CM-Asp, which can be
used to lift proteins from expression libraries to make protein
libraries from cells. The membranes also can be used for screening
of engineered enzymes. Application of the subject invention can
also be extended to purification of any interacting molecule, e.g.,
nucleic acids or small co-factors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] FIG. 1 is an outline illustrating a process for purifying
recombinant 6.times.His protein using CM-Asp chelating resin.
[0032] FIGS. 2A-2B show a comparison of Co.sup.2+ CM-Asp chelating
resin with Ni-NTA on 6.times.His prepro-.alpha.-factor purification
under denaturing conditions using pH gradient.
[0033] Legend for FIGS. 2A-2B: lane 1: crude lysate; lane 2:
flowthrough; lane 3: washed with 6 M Gu-HCl, 0.1 M
NaH.sub.2PO.sub.4, pH 8.0; lane 4: washed with 8 M urea, 0.1 M
NaH.sub.2PO.sub.4; lane 5: washed with 8 M urea, 0.1 M
NaH.sub.2PO.sub.4, pH 8.0; lane 6: deluted with 8 M urea, 0.1 M
NaH.sub.2PO.sub.4, pH 6.3; lane 7: deluted with 8 M urea, 0.1 M
NaH.sub.2PO.sub.4, pH 6.3; lane 8: deluted with 8 M urea, 0.1 M
NaH.sub.2PO.sub.4, pH 6.3; lane 9: deluted with 8 M urea, 0.1 M
NaH.sub.2PO.sub.4, pH 5.9; lane 10: deluted with 8 M urea, 0.1 M
NaH.sub.2PO.sub.4, pH 4.5; lane 11: deluted with 6 M Gu-HCl, 0.1 M
NaH.sub.2PO.sub.4, 0.2 M acetic acid; lane M: MW size markers.
[0034] FIG. 3 shows 6.times.His tagged DHFR purification by
Co.sup.2+ CM-Asp chelating resin under native conditions. Legend:
Lane 1: clarified lysate; lane 2: flowthrough; lane 3: first wash;
lane 4: third wash; lane 5: DHFR final elution.
[0035] FIG. 4 shows 6.times.His tagged DHFR purification by
Co.sup.2+ CM-Asp chelating resin under denaturing conditions.
Legend: Lane I: clarified lysate; lane 2: flowthrough; lane 3:
first pH 7.0 wash; lane 4: second pH 7.0 wash; lane 5: DHFR, first
pH 6.0 elution; lane 6: DHFR, second pH 6.0 elution.
[0036] FIG. 5 shows 6.times.His tagged DHFR purification by
Co.sup.2+ CM-Asp chelating resin under native conditions with
increasing concentrations of .beta.-mercaptoethanol. Legend: lane
1: 20 .mu.l of cell lysate; lanes 2, 4, 6, and 8: 20 .mu.l of
flowthrough; lanes 3, 5, 7, and 9: 5 .mu.l of eluant.
[0037] FIG. 6 shows yields of 6.times.His DHFR from cell lysates
purified by Co.sup.2+ CM-Asp chelating resin versus Ni-NTA in the
presence of .beta.-mercaptoethanol. Protein concentrations were
determined by Bradford assay. Yields are expressed as a percentage
of total protein in the cell lysate.
[0038] FIGS. 7A-7B show purification of 6.times.His GFP by
Co.sup.2+ CM-Asp chelating resin under native conditions. The GFP
bands were detected using Clontech's chemiluminescence Western
Exposure Kit and overnight exposure to x-ray film.
[0039] Legend: lane 1: clarified lysate; lane 2: flowthrough; lane
3: first wash; lane 4: first elution; lane 5: second elution; lane
6: third elution; lane 7: fourth elution.
[0040] FIG. 8 shows biological activity of 6.times.His GFP purified
by Co.sup.2+ CM-Asp chelating resin. Legend: tube 1: cell lysate;
tube 3: flowthrough, tube 3: wash, tube 4: first elution; tube 5:
second elution; tube 6: third elution.
DETAILED DISCLOSURE OF THE INVENTION
[0041] The subject method, which employs a CM-Asp metal chelating
complex, can advantageously be used for purification of recombinant
proteins having a polyhistidine tail or "tag."
[0042] According to one embodiment of the subject invention, a
resin ligand, e.g., CM-Asp, is complexed to a metal other than
Ca.sup.2+, forming a CM-Asp-metal complex. Preferably, the CM-Asp
ligand used in the subject invention is complexed with one of the
transition metals known as a third-block transition metal), e.g.,
Fe.sup.2+, Co.sup.2+, Ni.sup.2+, Cu.sup.2+, or Zn.sup.2+ in an
octahedral geometry. Other polymer matrices, e.g., polystyrene (as
in microtiter plates), nylon (as in nylon filters), SEPHAROSE
(Pharmacial, Uppeala, Sweden) or the like, can be used with the
subject invention and would readily be recognized by persons of
ordinary skill in the art. The poly-histidine tag possesses
"neighboring" histidine residues which can advantageously allow the
recombinant protein to bind to these transition metals in a
cooperative manner to form very strong metal ion complexes. This
cooperative binding refers to what is commonly known in the art as
a "neighboring histidine effect." For purposes of the subject
invention, and as would be understood by a person of ordinary skill
in the art, a "strong" or "very strong" metal ion complex refers to
the bond strength between the metal ion and the chelating ligand. A
strong or very strong metal ion complex, for example, allows little
or essentially no metal leakage from the complex so that the
purified protein, e.g., a recombinant protein having a
polyhistidine tag, is not contaminated with undesired or
"background" protein from a mixture being purified.
[0043] The CM-Asp metal complex offers two available valencies that
can form strong and reversible metal complexes with two adjacent
histidine residues on the surface of the recombinant protein
Another advantage to using the CM-Asp ligand is its ability to
strongly anchor the metal ion whereby metal ion leaking can be
virtually eliminated compared to metal leakage observed for other
complex binding agents, e.g., Ni-NTA. In a more preferred
embodiment, Co.sup.2+ can be used as the transition metal with
CM-Asp. The Co.sup.2+-CM-Asp can be less sensitive to reducing
agents, such as .beta.-mercaptoethanol. Metal ion leakage has been
shown to remain low, even negligible, in the presence of up to 30
mM .beta.-mercaptoethanol.
[0044] One embodiment of the purification process of the subject
invention is as follows: [0045] 1. Prepare lysate/sonicate
containing recombinant 6.times.His protein according to standard
procedures and techniques well known in the art. [0046] 2. Bind
6.times.His protein onto metal-loaded CM-Asp chelating resin at
slightly basic pH, e.g., about pH 8.0.
[0047] 3. Wash protein/resin complex at the same basic pH (about pH
8.0). Optional washes at a pH of about 7.0 or with imidazole
additive can also be included.
[0048] 4. Elute pure recombinant 6.times.His protein with an
elution buffer having a pH of about 6.0-6.3 or, in the alternative,
an elution buffer having a pH of about 8.0, plus about 40 to about
100 mM imidazole.
[0049] The steps involved in a preferred embodiment of the
purification process of the subject invention are illustrated in
FIG. 1. The subject process can be employed batchwise, in spin
columns, and in large-scale continuous-flow columns.
[0050] Buffers used in the above procedures are standard buffers
typically used in similar procedures, with appropriate adjustments
and modifications made as understood in the art For example, a high
ionic strength buffer, e.g., 50 mM phosphate/10 mM Tris/100 mM NaCl
can be used, with the pH adjusted as needed. The phosphate salt
component can range from a concentration of 10-100 mM; Tris from
5-25 mM; and NaCl from 50-200 mM.
[0051] Optimal elution conditions depend on the type of impurities,
the amount of protein to be purified, and unique properties of the
protein, and are determined on a case-by-case basis as would be
readily recognized by a person of ordinary skill in the art The
subject invention also pertains to a method for synthesizing
carboxymethylated aspartate chelating matrices, comprising the
steps of: [0052] (a) Michael addition of the .alpha.-amino function
of monoprotected .alpha.,.omega.-diamino acids to maleic acid;
[0053] (b) deprotecting the (.omega.-amino functionality; and
[0054] (c) attaching the chelator primary amine molecule to a solid
matrix.
[0055] Following are examples which illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
Large-Scale Preparation of CM-Asp Chelating Resin
[0056] SEPHAROSE CL-6B or CL-6B (Pharmacia, 8.0 L) is washed with
ddH.sub.2O, suction dried, and transferred to a 22-L round bottom
flask equipped with mechanical stirring. Epichlorohydrin (about 2.0
L) is added, the Sepharose resin mixed to a thick suspension, and
allowed to stand at room temperature for about 20 minutes. A
solution of sodium hydroxide (about 560 g) and sodium borohydride
(about 48 g) in approximately 6400 mL ddH.sub.2O is added and the
mixture stirred overnight at ambient temperature. The
oxirane-derivatized resin, collected by filtration, is washed ten
times with ddH.sub.2O (about 10 L each), once with 10% sodium
carbonate (about 10 L), suction dried, and transferred to a 22L
round bottom flask. A specimen of the oxirane derivatized resin
treated with 1.3 M sodium thiosulfate is titrated to approximately
pH 7.0 to determine the oxirane concentration (preferably, >700
.mu.mol/g).
[0057] To a solution of sodium hydroxide (approximately 268 g) in
about 7.6 L-ddH.sub.2O is added L-aspartic acid (about 575 g) and
sodium carbonate (about 1700 g), keeping the temperature below
about 25.degree. C. The pH is adjusted to approximately 11.0 and
the solution added to the resin. Using mechanical stirring and a
heating mantle, the reaction mixture is brought to about 80.degree.
C. for 4 hours and allowed to cool to room temperature overnight.
The resin was collected by filtration, washed ten times with
ddH.sub.2O (about 10 L each), once with 10% sodium carbonate (about
10 L), suction dried, and transferred to a 22-L round bottom flask
equipped with mechanical stirring.
[0058] To an ice-cooled solution of sodium hydroxide (about 900 g)
in 12 L ddH.sub.2O was added bromoacetic acid (about 3000 g) in
approximately 750 g increments, keeping temperature below about
30.degree. C. Sodium carbonate (about 660 g) is added and the pH is
adjusted to about 10. The resin is reacted with the solution at
ambient temperature overnight The resin is collected by filtration,
washed six times with ddH.sub.2O (about 10 L each), six times with
10% acetic acid, and ten times with ddH.sub.2O. Washing is
continued with ddH.sub.2O until the pH reached about 6.0 by litmus
paper. The CM-Asp chelating resin was suction dried in preparation
for metal loading.
EXAMPLE 2
Preparation of C-Linked CM-Asp Chelating Resin
[0059] N.sup.6-Carbobenzyloxy-L-lysine (6.15 g) and excess maleic
acid (17.6 g) were dissolved in 2 M NaOH (35 mL). The solution was
refluxed for 24 hr and allowed to cool to ambient. The pH was
adjusted to 3 with 6 M HCl and chilled. The mixture was filtered to
remove the unreacted maleic acid which was washed with water (15
mL). The filtrate and washings were combined and evaporated. The
residue was dissolved in 4% ammonium formate (120 mL) and degassed
briefly. 10% Pd/C (600 mg) was added and the mixture was refluxed
under Ar with stirring for 5 hr. The mixture was filtered through
celite and the filtrate evaporated. The residue was dissolved in a
solution of sodium hydroxide (1.3 g) and sodium carbonate (8.7 g)
in dd H.sub.2O (40 mL). The final pH was adjusted to 11. This
solution is added to the oxirane-derivatized resin (40 mL bed
volume) prepared as described in Example 1. The mixture was stirred
at 60.degree. with mechanical stirring for 4 hr and at ambient for
16 hr. The resin was collected by filtration, washed with
ddH.sub.2O (6.times.100 mL), 10% HOAc (6.times.100 mL), and
ddH.sub.2O (6.times.100 mL) or until the pH reached about 6 by
litmus paper test The C-Linked CM-Asp chelating resin was suction
dried in preparation for metal loading.
EXAMPLE 3
Metal Loading of CM-Asp Chelating Resin
[0060] The CM-Asp chelating resin of Example 1 (about 1 L of
suction dried bed volume) is treated with a transition metal ion
solution, e.g., 2 L of either 200 mM of cobalt chloride
hexahydrate, nickel sulfate hexahydrate, copper sulfate
pentahydrate, or zinc chloride, according to the metal ion
deserved. The resin is reacted with the 200 mM metal solution at
room temperature for approximately 72 hours and then collected by
filtration. The metal loaded CM-Asp chelating resin is washed five
times with ddH.sub.2O (about 1 L each), two times with 100 mM NaCl
(about 1 L each), six times with ddH.sub.2O (about 1 L each), and
once with 20% aq. ethanol (about 1 L). The resin can be stored in
20% aq. ethanol.
EXAMPLE 4
Comparison of Co.sup.2+ CM-Asp Resin With Ni.sup.2+-NTA on
Recombinant 6.times.His Prepro-.alpha.-Factor Under Denaturing
Conditions Using pH Gradient
[0061] For a qualitative comparison of the purification of
Co.sup.2+ CM-Asp chelating resin and Ni-NTA under denaturing
conditions, the C-terminal, 6.times.His-tagged
prepro-.alpha.-factor of S. cerevisiae was expressed in E. coli.
One gram bacterial cell pellet was lysed in 6 M guanidinium-HCl
(Gu-HCl) and 0.1 M NaH.sub.2PO.sub.4, pH 8.0. Three milliliters of
clarified lysate was loaded onto a Co.sup.2+ CM-Asp chelating resin
gravity flow column. The resin-proteins mixture was washed with 8 M
urea, 0.1 M NaH.sub.2PO.sub.4, pH 8.0, and deluted with 8 M urea,
0.1 M NaH.sub.2PO.sub.4 at three different pHs, 6.3, 5.9, and 4.5.
Finally, all bound proteins were deluted with 6 M Gu-HCl, 0.1 M
NaH.sub.2PO.sub.4, 0.2 M acetic acid. Samples from each step were
loaded on a 12% polyacrylamide/SDS gel, electrophoresed, and the
gel was stained with Coomassie blue. The 6,His-tagged
prepro-.alpha.-factor was deluted at pH 6.3 as a single prominent
band on the gel.
[0062] In the same manner, 3 ml of clarified lysate was loaded onto
a Ni-NTA gravity flow column. The resin-proteins mixture was washed
and deluted the same as above. Samples from each step were loaded
on a 12% polyacrylamide/SDS gel, electrophoresed, and the gel was
stained with Coomassie blue. There were more than 10 protein bands
in elution at pH 6.3. The 6.times.His-tagged prepro-.alpha.-factor
was a minor band among them. The majority of the protein was
deluted at pH 4.5 without any other contaminant proteins. This
demonstrated that the highly purified 6.times.His-tagged
prepro-.alpha.-factor was deluted from Co.sup.2+ CM-Asp chelating
resin at the conditions (pH 6.3) under which Ni-NTA was still
releasing contaminants. The affinity of Co.sup.2+ CM-Asp chelating
resin to 6.times.His-tagged prepro-.alpha.-factor was more
selective than Ni-NTA to the protein.
[0063] Results show that highly purified 6.times.His-tagged protein
elutes from Co.sup.2+ CM-Asp chelating resin while Ni-NTA is still
releasing contaminants. See FIGS. 2A-2B: FIG. 2A: Results after
using 1 ml of Co.sup.2+ CM-Asp chelating resin. FIG. 2B: Results
after using 1 ml of nickel-NTA.
EXAMPLE 5
Recombinant 6.times.His DHFR Purification with CM-Asp Resin Under
Native Conditions
[0064] N-terminal, 6.times.His-tagged mouse dihydrofolate reductase
(DHFR, MW 20.3 kDa) was expressed in E. coli cells. Cells were then
pretreated with 0.75 mg/ml lysozyme and disrupted in lysis buffer
(100 mM NaH.sub.2PO.sub.4, 10 mM Tris-HCl, pH 8.0) by mechanical
shearing, 800 .mu.l of the clarified lysate was applied to 100
.mu.l of Co.sup.2+ CM-Asp chelating resin, pre-equilibrated with
lysis buffer, and washed with one ml of lysis buffer three times.
All bound protein was deluted by 300 .mu.l of 100 mM EDTA, pH 8.0.
Twenty microliters of lysate and 40 .mu.l of each subsequent
fraction from elution were run on a 12% polyacrylamide/SDS gel. The
gel was stained with Coomassie blue. One single protein band was
shown at a position of MW 20.3 kDa Results showed the selective
binding affinity of Co.sup.2+ CM-Asp chelating resin to
6.times.His-tagged DHFR under native purification conditions. No
discernable binding of host proteins occurred.
[0065] Results show that Co.sup.2+ CM-Asp chelating resin has
selective binding affinity for 6.times.Histidines. No discernable
binding of host proteins occurred. See FIG. 3.
EXAMPLE 6
Recombinant 6.times.His DHFR Purification with CM-Asp Resin Under
Denaturing Conditions
[0066] N-terminal 6.times.His-tagged mouse DHFR was expressed in a
25-ml culture of E. coli. Cells were pelleted, resuspended in lysis
buffer (100 mM NaH}PO.sub.4, 10 mM Tris-HCl, 8 M urea, pH 8.0), and
disrupted by vortexing. Six hundred microliters of clarified lysate
were applied to a Co.sup.2+ CM-Asp chelating resin spin column
containing 0.5 ml of Co.sup.2+ CM-Asp chelating resin-NX metal
affinity resin and centrifuged for 2 minutes at 2,000.times.g. The
column was washed twice with 1 ml of wash buffer (100 mM
NaH.sub.2PO.sub.4, 10 mM PIPES, pH 7.0), and bound proteins were
deluted with 600 .mu.l of elution buffer (20 mM PIPES, 100 mM NaCl,
8 M urea, pH 6.0). Twenty microliters of lysate and 40 .mu.l of
each subsequent fraction from the elution were loaded onto a 12%
polyacrylamide/SDS gel and electrophoresed. The gel was stained
with Coomassie blue. One single protein band was shown at the
position of 20.3 kDa Results showed the selective binding affinity
of Co.sup.2+ CM-Asp chelating resin to 6.times.His-tagged DHFR
under denaturing conditions. The binding properties of Co.sup.2+
CM-Asp chelating resin to 6.times.histidines allow proteins deluted
under mild pH conditions (pH 6.0) that protect protein
integrity.
[0067] Results show that bound protein can be deluted at mild pH
(pH 6.0). This indicates that the binding properties of Co.sup.2+
CM-Asp chelating resin allow protein elution under mild pH
conditions that protect protein integrity. See FIG. 4.
EXAMPLE 7
Recombinant 6.times.His DHFR Purification with CM-Asp Resin Under
Native Conditions with Increasing Concentrations of
Beta-Mercaptoethanol
[0068] N-terminal, 6.times.His-tagged mouse DHFR was expressed in
E. coli. Twenty-five milliliters of cell culture were disrupted in
2 ml of sonication buffer (100 mM NaH.sub.2PO.sub.4, 10 mM
Tris-HCl, and 100 mM NaCl, pH 8.0) by freezing and thawing. Then,
2.66 ml of clarified lysate were applied to a 200-.mu.l Co.sup.2+
CM-Asp chelating resin gravity flow column, pre-equilibrated with
the sonication buffer. The proteins/resin mixtures were washed
three times with sonication buffer, pH 8.0. All bound proteins were
deluted with 600 .mu.l of 100 mM EDTA, pH 8.0. To test the effect
of .beta.-mercaptoethanol on the Co.sup.2+ CM-Asp chelating resin
purification under native conditions, all buffers used here
contained either 0, 10, 20, or 30 mM .beta.-mercaptoethanol.
Samples from each elution were electrophoresed on a 12%
polyacrylamide/SDS gel and the gel was stained with Coomassie blue.
One single protein band at the position of MW 20.3 kDa was shown
from all elutions. The presence of .beta.-mercaptoethanol did not
obsolete the purity of 6.times.His-tagged DHFR purified by
Co.sup.2+ CM-Asp chelating resin. With up to 30 mM
.beta.-mercaptoethanol in all purification buffers, there was no
predominant band at 20.3 kDa in flowthroughs, indicating that no
loss of metal occurred during protein purification using Co.sup.2+
CM-Asp chelating resin in the presence of
.beta.-mercaptoethanol.
[0069] Results show that with up to 30 mM .beta.-mercaptoethanol in
the purification buffer, there is no predominant band at 20.3 kDa
in the flowthrough, indicating that no loss of metal occurred
during protein purification using Co.sup.2+ CM-Asp chelating resin
in the presence of .beta.-mercaptoethanol. See FIG. 5.
EXAMPLE 8
Yields of 6.times.His DHFR From Cell Lysates Purified by CM-Asp
Versus Ni-NTA in the Presence of Beta-Mercaptoethanol
[0070] N-terminal, 6.times.His-tagged DHFR was expressed and
purified by Co.sup.2+ CM-Asp chelating resin under native
conditions as described in Example 7. Protein concentrations were
determined by Bradford assay. Yields were expressed as a percentage
of total protein in the cell lysate. The yields of purified
6.times.His-tagged DHFR were 14%, 28%, 34%, and 35% respectively,
with .beta.-mercaptoethanol present in purification buffers at the
concentrations of 0, 10, 20, and 30 mM. The protein was purified by
Ni-NTA under the same native conditions; the yields of purified
6.times.His-tagged DHFR were 4%, 8.8%, 3.4%, and 4% respectively
with .beta.-mercaptoethanol present in purification buffers at the
concentrations of 0, 10, 20, and 30 mM. The yields of purified
6.times.His-tagged DHFR were significantly higher when using
Co.sup.2+ CM-Asp chelating resin compared to Ni-NTA under native
purification conditions with .beta.-mercaptoethanol. This indicates
that the metal ion on Co.sup.2+ CM-Asp chelating resin is strongly
anchored to SEPHAROSE beads by a CM-ASP metal chelator that is
ideal for binding octahedral metals.
[0071] Results show that the yields of purified 6.times.His DHFR
are significantly higher at 10, 20, and 30 mM
.beta.-mercaptoethanol using Co.sup.2+ CM-Asp chelating resin
compared to using Ni-NTA. This indicates that the metal ion on
Co.sup.2+ CM-Asp chelating resin is strongly anchored to sepharose
beads by CM-Asp metal chelator that is advantageous for binding
octahedral metals. See FIG. 6.
EXAMPLE 9
Purification of 6.times.His GFP by Co.sup.2+-CM-Asp Under Native
Conditions
[0072] N-terminal, 6.times.His-tagged green fluorescent protein
(GFP) was expressed in E. coli cells. Cells were pelleted,
resuspended in sonication buffer(100 mM NaH.sub.2PO.sub.4, 10 mM
Tris-HCl, and 100 mM NaCl, pH 8.0), and disrupted by freezing and
thawing three times. Two milliliters of clarified lysate were
applied to 400 .mu.l of Co.sup.2+ CM-Asp chelating resin,
pre-equilibrated with sonication buffer, and washed three times
with 2 ml of sonication buffer, pH 8.0. The 6.times.His-tagged GFP
was deluted with 400 .mu.l of 75 mM imidazole buffer containing 20
mM Tris-HCl and 100 mM NaCl, pH 8.0. Samples from each purification
step were loaded onto a 12% polyacrylamide/SDS gel,
electrophoresed, and the gel was stained with Coomassie blue. One
single band was shown at the position of MW 27.8 kDa in the elution
with 75 mM imidazole. This demonstrated that 6.times.His-tagged GFP
selectively bound on Co.sup.2+ CM-Asp chelating resin and can be
deluted with low concentration of imidazole under native
purification conditions.
[0073] Samples from each purification step were also loaded on a
12% polyacrylamide/SDS gel, electrophoresed, and transblotted to a
PVDF membrane. The proteins on the blot were probed with anti-GFP
monoclonal antibody. One single GFP band was clearly shown in the
samples of cell lysate and elution. There was no GFP band shown in
flowthrough, which indicated that all expressed GFP in cell lysate
was bound to Co.sup.2+ CM-Asp chelating resin.
[0074] Results show that (FIG. 7A) Coomassie blue stained gel shows
one single band in 75 mM imidazole elution. This indicates that
6.times.Histidines selectively bound on Co.sup.2+ CM-Asp chelating
resin. Western analysis data shows no GFP in flowthrough which
indicates the high affinity between Co.sup.2+ CM-Asp chelating
resin and 6.times.histidines (FIG. 7B).
EXAMPLE 10
Biological Activity of 6.times.His GFP Purified by Co2+ CM-Asp
[0075] N-terminal, 6.times.His-tagged GFP was expressed in E. coli.
Cell lysate was prepared as described in Example 7. The cell lysate
was loaded onto a 2-ml Co.sup.2+ CM-Asp chelating resin Disposable
Gravity Column, and purified using the Batch/Gravity Flow column
purification method as described in Example 9. The column was
washed with sonication buffer three times and deluted with 100 mM
EDTA, pH 8.0. Samples were collected in microfuge tubes from each
purification step. The fluorescence of all collected samples was
detected using an UltraLum Electronic U.V. Transilluminator.
Samples of cell lysate and elution showed strong fluorescence. This
experiment demonstrated that 6.times.His-tagged GFP can be purified
to homogeneity by Co.sup.2+ CM-Asp chelating resin under native
conditions and maintains biological activity.
[0076] The photo of samples from each purification step shows that
GFP can be purified to homogeneity by Co.sup.2+ CM-Asp chelating
resin under native conditions, and the fluorescence indicates that
GFP purified by Co.sup.2+ CM-Asp chelating resin still maintains
its biological activity. See FIG. 8.
[0077] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims.
* * * * *