U.S. patent application number 12/019985 was filed with the patent office on 2008-08-14 for methods for separating molecules.
This patent application is currently assigned to PROMEGA CORPORATION. Invention is credited to Rebecca Godat, Tonny Johnson, Sanchayita Kar, John Schultz, Daniel J. Simpson.
Application Number | 20080193996 12/019985 |
Document ID | / |
Family ID | 32108114 |
Filed Date | 2008-08-14 |
United States Patent
Application |
20080193996 |
Kind Code |
A1 |
Simpson; Daniel J. ; et
al. |
August 14, 2008 |
METHODS FOR SEPARATING MOLECULES
Abstract
The present invention provides compositions and methods for the
separation of metals or molecules such as polypeptides, nucleic
acids, or endotoxins using a metal-modified solid support. The
metals or molecules are isolated from a starting material using the
modified solid supports of the invention. Also provided by the
invention are kits that can be used in connection with the
inventive methods.
Inventors: |
Simpson; Daniel J.;
(Middleton, WI) ; Johnson; Tonny; (Madison,
WI) ; Schultz; John; (Verona, WI) ; Godat;
Rebecca; (DeForest, WI) ; Kar; Sanchayita;
(Madison, WI) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH LLP
ONE SOUTH PINCKNEY STREET, P O BOX 1806
MADISON
WI
53701
US
|
Assignee: |
PROMEGA CORPORATION
Madison
WI
|
Family ID: |
32108114 |
Appl. No.: |
12/019985 |
Filed: |
January 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10689368 |
Oct 20, 2003 |
7354750 |
|
|
12019985 |
|
|
|
|
60419614 |
Oct 18, 2002 |
|
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Current U.S.
Class: |
435/174 ;
530/385; 530/400; 530/412 |
Current CPC
Class: |
B01J 20/3234 20130101;
B01J 20/3219 20130101; B01J 20/3251 20130101; B01D 15/3828
20130101; B01J 20/3204 20130101; B01J 20/3259 20130101; B01J
20/3293 20130101; C02F 1/285 20130101; B01J 20/286 20130101; C02F
2101/20 20130101; B01J 45/00 20130101; C07K 1/20 20130101; B01J
20/3242 20130101; B01J 2220/58 20130101; B01J 20/3289 20130101;
B01J 2220/66 20130101; B01J 20/3217 20130101; B01J 20/28009
20130101; B01J 20/103 20130101; B01J 20/3265 20130101; G01N
2030/042 20130101 |
Class at
Publication: |
435/174 ;
530/412; 530/385; 530/400 |
International
Class: |
C12N 11/00 20060101
C12N011/00; C07K 14/00 20060101 C07K014/00; C07K 14/805 20060101
C07K014/805 |
Claims
1. A method for isolating membrane-associated proteins from a
starting material comprising (a) contacting the starting material
with a composition under suitable conditions to form a complex
between the membranes and the composition, wherein the starting
material comprises an in vitro expression system comprising
microsomal membrane vesicles, and wherein the composition
comprises: ##STR00005## wherein X is a substituted or unsubstituted
alkylene moiety, a substituted or unsubstituted aralkylene moiety,
or a substituted or unsubstituted arylene moiety; R.sub.1 is a
hydrocarbon moiety, or a substituted hydrocarbon moiety; R.sub.2
and R.sub.3 are independently selected from R.sub.1, a hydrocarbon
moiety, a substituted hydrocarbon moiety, a halogen atom, a
hydrogen atom, a hydroxy, a thiol, an amine, a silanol bond to the
solid support, a bond to another silane ligand, or
O--Si--Y.sub.1Y.sub.2Y.sub.3, wherein Y.sub.1, Y.sub.2 and Y.sub.3
are independently selected from a hydrocarbon moiety, or a
substituted hydrocarbon moiety; and R.sub.N is NH.sub.2,
NHR.sub.N1, NR.sub.N1R.sub.N2, or NR.sub.N1R.sub.N2R.sub.N3,
wherein R.sub.N1, R.sub.N2, and R.sub.N3 are independently selected
from a hydrocarbon moiety, a substituted hydrocarbon moiety, or a
hydrogen atom.
2. The method of claim 1, wherein R.sub.N1, R.sub.N2, and R.sub.N3
are independently selected from the group consisting of an alkyl
moiety of up to six carbon atoms in a longest chain, a substituted
alkyl moiety of up to six carbon atoms in a longest chain, and a
hydrogen atom.
3. The method of claim 1, further comprising contacting the complex
of step (a) with a nonionic detergent.
4. A method for separating target material from non-target material
in a starting material comprising: (a) contacting the starting
material with a composition to form a complex between the target
material and the composition, wherein the composition comprises:
##STR00006## wherein X is a substituted or unsubstituted alkylene
moiety, a substituted or unsubstituted aralkylene moiety, or a
substituted or unsubstituted arylene moiety; R.sub.1 is a
hydrocarbon moiety, or a substituted hydrocarbon moiety; R.sub.2
and R.sub.3 are independently selected from R.sub.1, a hydrocarbon
moiety, a substituted hydrocarbon moiety, a halogen atom, a
hydrogen atom, a hydroxy, a thiol, an amine, a silanol bond to the
solid support, a bond to another silane ligand, or
O--Si--Y.sub.1Y.sub.2Y.sub.3, wherein Y.sub.1, Y.sub.2 and Y.sub.3
are independently selected from a hydrocarbon moiety, or a
substituted hydrocarbon moiety; and R.sub.N is NH.sub.2,
NHR.sub.N1, NR.sub.N1R.sub.N2, or NR.sub.N1R.sub.N2R.sub.N3,
wherein R.sub.N1, R.sub.N2, and R.sub.N3 are independently selected
from a hydrocarbon moiety with up to a six-carbon main chain, a
substituted hydrocarbon moiety with up to a six-carbon main chain,
or a hydrogen atom; wherein at least a portion of the non-target
material does not form a complex with the composition, wherein the
non-target material comprises a polypeptide containing a prosthetic
heme group.
5. The method of claim 4, wherein R.sub.N1, R.sub.N2, and R.sub.N3
are independently selected from the group consisting of an alkyl
moiety of up to six carbon atoms in a longest chain, a substituted
alkyl moiety of up to six carbon atoms in a longest chain, and a
hydrogen atom.
6. The method of claim 4, wherein the non-target material is
selected from the group consisting of hemoglobin and myoglobin.
7. A method for removing cells from a starting material comprising:
(a) contacting the starting material with a composition under
conditions suitable to form a complex between the cells and the
composition, the composition comprising: ##STR00007## wherein X is
a substituted or unsubstituted alkylene moiety, a substituted or
unsubstituted aralkylene moiety, or a substituted or unsubstituted
arylene moiety; R.sub.1 is a hydrocarbon moiety, or a substituted
hydrocarbon moiety; R.sub.2 and R.sub.3 are independently selected
from R.sub.1, a hydrocarbon moiety, a substituted hydrocarbon
moiety, a halogen atom, a hydrogen atom, a hydroxy, a thiol, an
amine, a silanol bond to the solid support, a bond to another
silane ligand, or O--Si--Y.sub.1Y.sub.2Y.sub.3, wherein Y.sub.1,
Y.sub.2 and Y.sub.3 are independently selected from a hydrocarbon
moiety, or a substituted hydrocarbon moiety; and R.sub.N is
NH.sub.2, NHR.sub.N1, NR.sub.N1R.sub.N2, or
NR.sub.N1R.sub.N2R.sub.N3, wherein R.sub.N1, R.sub.N2, and R.sub.N3
are independently selected from a hydrocarbon moiety, a substituted
hydrocarbon moiety, or a hydrogen atom.
8. The method of claim 7, wherein R.sub.N1, R.sub.N2, and R.sub.N3
are independently selected from the group consisting of an alkyl
moiety of up to six carbon atoms in a longest chain, a substituted
alkyl moiety of up to six carbon atoms in a longest chain, and a
hydrogen atom.
9. The method of claim 7, wherein R.sub.N1, R.sub.N2, and R.sub.N3
are independently selected from an alkyl moiety of up to six carbon
atoms in a longest chain, a substituted alkyl moiety of up to six
carbon atoms in a longest chain, and a hydrogen atom.
10. The method of claim 7, wherein the conditions comprise the
presence of methanol, ethanol, or isopropanol in a concentration of
at least about 1% (v/v).
11. The method of claim 7, wherein the conditions comprise the
presence of isopropanol in a concentration of at least about 15%
(v/v).
12. The method of claim 7, wherein the cells are bacterial cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/689,368, filed Oct. 20, 2003, which claims priority to U.S.
Provisional Application No. 60/419,614, filed Oct. 18, 2002, which
are incorporated herein by reference in their entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
BACKGROUND OF THE INVENTION
[0003] The present invention relates broadly to compositions and
methods for separating metal ions or other target material,
including, but not limited to, polypeptides, nucleic acids, or
endotoxins, from non-target material.
SUMMARY OF THE INVENTION
[0004] In one aspect, the invention includes methods for isolating
target material from a starting material comprising contacting the
starting material with a composition selected from the group
consisting of:
##STR00001##
wherein R.sub.1 is
##STR00002##
[0005] X is a substituted or unsubstituted alkylene moiety, a
substituted or unsubstituted aralkylene moiety, or a substituted or
unsubstituted arylene moiety;
[0006] R.sub.2 and R.sub.3 are independently selected from R.sub.1,
a hydrocarbon moiety, a substituted hydrocarbon moiety, a halogen
atom, a hydrogen atom, a hydroxy, a thiol, an amine, a silanol bond
to the solid support, a bond to another silane ligand, or
O--Si--Y.sub.1Y.sub.2Y.sub.3, wherein Y.sub.1, Y.sub.2 and Y.sub.3
are independently selected from a hydrocarbon moiety or a
substituted hydrocarbon moiety;
[0007] R.sub.4 is a hydrocarbon moiety, a substituted hydrocarbon
moiety, or a hydrogen atom;
[0008] M.sup.+ is a metal ion; and
[0009] n is an integer.gtoreq.1;
and
##STR00003##
wherein X is a substituted or unsubstituted alkylene moiety, a
substituted or unsubstituted aralkylene moiety, or a substituted or
unsubstituted arylene moiety;
[0010] R.sub.4 is a hydrocarbon moiety, a substituted hydrocarbon
moiety, or a hydrogen atom;
[0011] M.sup.+ is a metal ion;
[0012] n is an integer.gtoreq.1; and
[0013] m is 0 or 1;
to form a complex between at least a portion of the target material
and the composition.
[0014] These and other aspects of the present invention will be
better appreciated by reference to the following drawings and
Detailed Description.
[0015] Each of the publications or patent applications cited herein
is incorporated by reference in its entirety. In the case of
conflict between the present disclosure and an incorporated
publication, the present disclosure should control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 provides graphs comparing fractionation of
hemoglobin, alkaline phosphatase, and B-galactosidase on
aminopropyl-modified magnetic silica particles (FIG. 1A),
Q-sepharose resin (FIG. 1B), and DEAE resin (FIG. 1C).
[0017] FIG. 2 is an SDS-PAGE gel of FluoroTect-Green labeled
luciferase and hemoglobin fractionated using aminopropyl-modified
magnetic silica particles.
[0018] FIGS. 3A and 3B are SDS-PAGE gels of glycosylated membrane
proteins captured by 3-aminopropyl magnetic silica particles.
[0019] FIG. 4 is an SDS-PAGE gel of membrane proteins expressed in
cell-free expression systems isolated using 3-aminopropyl magnetic
silica particles.
[0020] FIG. 5 is a graph illustrating capture of bacterial cells by
3-aminopropyl-modified magnetic silica particles.
[0021] FIG. 6 is an SDS-PAGE gel of his-tagged RNase HI purified
using nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0022] FIG. 7 is an SDS-PAGE gel comparing fractionation of
hemoglobin using various metal
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0023] FIG. 8 is an SDS-PAGE gel of his-tagged RNase HI
fractionated using aminopropyl-modified magnetic silica particles
followed by nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0024] FIG. 9A is a photograph showing that Coomasie blue dye binds
to his-tagged luciferase attached to nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. FIG. 9B is a photograph showing elution of Coomasie
blue-stained his-tagged luciferase from nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles with increasing concentrations of imidazole. FIG. 9C is a
photograph comparing the Coomasie blue dye binding to his-tagged
proteins attached to nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles with that of other metal chelating resins.
[0025] FIG. 10A shows an SDS-PAGE gel of fluorescently labeled
his-tagged luciferase following fractionation by nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. FIG. 10B is an SDS-PAGE gel of fluorescently labeled
his-tagged BSA following fractionation by
copper-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic
silica particles.
[0026] FIG. 11 is an SDS-PAGE gel of tRNA isolated using nickel
(II) 3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic
silica particles.
[0027] FIG. 12 is an SDS-PAGE gel of his-tagged proteins from a
cell-free expression system isolated using nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0028] FIG. 13 is a graph comparing enzyme activity of free tRNA
synthetase with that of tRNA synthetase bound to nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0029] FIG. 14 shows pipette tips modified to include a solid
support according to the present invention for use in purification
and analysis of polypeptides.
[0030] FIG. 15 is an SDS-PAGE gel illustrating binding and elution
patterns of complex protein mixtures from rabbit reticulocyte
lysate using various metal
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0031] FIG. 16A is an SDS-PAGE gel illustrating binding and elution
patterns of complex protein mixtures from rabbit reticulocyte
lysate using copper
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. FIG. 16B is a graph showing protein concentrations
(A.sub.595 by Bradford method) of various fractions.
[0032] FIG. 17A is an SDS-PAGE gel showing binding and elution
patterns of complex protein mixtures from CHO cell lysate using
copper 3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic
silica particles. FIG. 17B is a graph showing protein
concentrations (A.sub.595 by Bradford method) of various
fractions.
[0033] FIG. 18A is an SDS-PAGE gel illustrating the binding and
elution patterns of complex protein mixtures from wheat germ cell
lysate using copper
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. FIG. 18B is a graph showing protein concentrations
(A.sub.595 by Bradford method) of various fractions.
[0034] FIG. 19 is an SDS-PAGE gel illustrating sequential elution
of proteins with copper
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles under various conditions.
[0035] FIG. 20 is an SDS-PAGE gel illustrating sequential elution
of proteins with copper
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles under various conditions.
[0036] FIG. 21 is an SDS-PAGE gel of the phosphoprotein ovalbumin
isolated using iron(III) or gallium (III)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0037] FIG. 22 is an SDS-PAGE gel of phosphoproteins isolated from
rabbit reticulocyte lysate using iron(III) or gallium (III)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles.
[0038] FIG. 23A is a photograph of Coomassie-stained His-tagged
proteins from bacterial lysates recovered at various times post
induction isolated using nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles; FIG. 23B is a graph of cell growth (as measured by
OD.sub.600) and protein concentration (as measured by A.sub.595 of
Coomassie stained proteins) as a function of time post-induction;
FIG. 23C is an SDS-PAGE gel of the purified protein in the
samples.
[0039] FIG. 24 shows a fluorescent image of an SDS-PAGE gel of
his-tagged proteins isolated using nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles and stained with BODIPY.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention provides compositions and methods for
separating target material from a starting material. Suitable
target material includes, but is not limited to, metal ions,
polypeptides, nucleic acids, whole cells, cell membranes, and the
like. The invention also provides kits suitable for use in the
practice of the methods of the invention.
[0041] The compositions and methods of the invention are useful in
a wide variety of applications, including, for example,
fractionation of molecules in a mixture, removing metal ions from a
fluid, capturing cells from a cell suspension, isolating membranes
or membrane proteins, removing undesired contaminating proteins
from a mixture, and the like. As one skilled in the art will
appreciate, the compositions and methods of the invention may be
used alone, or in conjunction with other compositions or
methods.
[0042] Because the compositions and methods of the invention permit
facile isolation or purification of target material, they are
susceptible to miniaturization, robotic manipulation, and use in
high throughput assays. The compositions and the methods of the
present invention are also suitable for use in structural and
functional genomics, or proteomics.
[0043] The compositions of the present invention comprise modified
solid supports, including nitrilotriacetic acid (NTA)-modified
solid supports and metal-modified solid supports.
[0044] The methods of the invention employ NTA-modified solid
supports, metal-modified solid supports, or amine-modified solid
supports to form a complex with, or otherwise effect separation of,
a target material such as metal ions, molecules, subcellular
components, or whole cells, from non-target material in a starting
material.
[0045] Suitable solid supports for making the modified solid
supports of the present invention include, without limitation, gels
or hard support material, agarose, polyacrylamide, cellulose,
plastics, polysaccharides, nylon, polystyrene, latex methacrylate,
silica, aluminum oxide, electrodes, membranes, and derivatives
thereof.
[0046] Suitable silica solid supports include, but are not limited
to, siliceous oxide, magnetic silica particles, solid silica such
as glass or diatomaceous earth and the like, or a combinations of
silica materials (see, e.g., preparation of silica discussion in
Kurt-Othmer Encyclopedia of Chemical Technology, Vol. 21, 4th ed.,
Mary Howe-Grant, ed., John Wiley & Sons, pub., 1997, p. 1021).
As discussed in the examples below, suitable silica gels are
available commercially from suppliers such as Silicycle (Quebec
City, Canada), J. T. Baker (Phillipsberg, N.J.), and Sigma-Aldrich,
(St. Louis, Mo.). Suitable silica gels for the compositions and
methods of the invention are further described in the examples
below. Other suitable silica supports include crystalline or
vitreous silicas, such as quartz, vitreous silica, controlled pore
glass particles, and glass fibers.
[0047] Silica gel may be characterized by pore diameter, particle
size, or specific surface area. Suitable silica gels have a pore
diameter from about 30 to about 1000 Angstroms, a particle size
from about 2 to about 300 microns, and a specific surface area from
about 50 m.sup.2/g to about 1000 m.sup.2/g. Suitable silica gels
include, for example, those having a pore diameter of about 40
Angstroms, about 60 Angstroms, and about 150 Angstroms; those
having a particle size of about 2 to about 25 microns, about 5 to
about 25 microns, about 15 microns, about 63 to about 200 microns
and about 75 to about 200 microns; and those having a specific
surface area of about 300 m.sup.2/g, about 500 m.sup.2/g, about 550
m.sup.2/g, about 675 m.sup.2/g, and about 750 m.sup.2/g.
[0048] Conveniently, a solid support according to the present
invention may comprise magnetic silica particles. Magnetic silica
particles comprise a superparamagnetic core coated with a hydrous
siliceous oxide adsorptive surface (i.e. a surface having silanol
or Si--OH groups). Suitable, commercially available magnetic silica
particles include MagneSil.TM. particles available from Promega
Corporation (Madison, Wis.). The preparation of magnetic silica
particles suitable for use as a support according to the present
invention is described in U.S. Pat. No. 6,296,937.
[0049] Suitable cellulose supports include, but are not limited to,
nitrocellulose and cellulose acetate.
[0050] Suitable membranes include, but are not limited to, glass
fiber membranes impregnated with silica.
[0051] Suitable aluminum oxide solid supports include, but are not
limited to, Brockmann aluminum oxides that are about 150 mesh and
58 angstroms.
[0052] An amine-modified solid support, as described herein, may be
formed, for example, using a solid support that includes at least
one free hydroxyl group such that, when the solid support is
contacted with an aminosilane compound, the silicon atom of the
aminosilane compound is covalently bound to the solid support by at
least one silanol bond to form an amine-modified solid support.
[0053] Aminosilane compounds are commercially available through
suppliers such as United Chemical Technologies, Inc. (Bristol,
Pa.). Suitable aminosilane compounds comprise
##STR00004##
wherein X is an alkylene moiety of up to 20 carbon atoms that may
be saturated, unsaturated, branched, linear, or cyclic, for
example, methylene, ethylene propylene, nonylene, or an aralkylene
moiety of up to 20 carbon atoms in which the alkyl portion may be
saturated, unsaturated, branched, linear, or cyclic or an arylene
moiety of up to 20 carbon atoms, and wherein X may be unsubstituted
or substituted as defined below with respect to hydrocarbon
moiety;
[0054] R.sub.1 is a hydrocarbon moiety, or a substituted
hydrocarbon moiety;
[0055] R.sub.2 and R.sub.3 are independently selected from R.sub.1,
a hydrocarbon moiety, a substituted hydrocarbon moiety, a halogen
atom, a hydrogen atom, a hydroxy, a thiol, an amine, a silanol bond
to the solid support, a bond to another silane ligand, or
O--Si--Y.sub.1Y.sub.2Y.sub.3, wherein Y.sub.1, Y.sub.2 and Y.sub.3
are independently selected from a hydrocarbon moiety, or a
substituted hydrocarbon moiety; and
[0056] R.sub.N is NH.sub.2, NHR.sub.N1, NR.sub.N1R.sub.N2, or
NR.sub.N1R.sub.N2R.sub.N3, wherein R.sub.N1, R.sub.N2, and R.sub.N3
are independently selected from a hydrocarbon moiety, a substituted
hydrocarbon moiety, or a hydrogen atom; suitably R.sub.N1,
R.sub.N2, and R.sub.N3 may independently be an alkyl moiety of up
to six carbon atoms in a longest chain, a substituted alkyl moiety
of up to six carbon atoms in a longest chain, or a hydrogen atom. A
"longest chain" is the longest chain of an alkyl moiety as utilized
in IUPAC nomenclature.
[0057] The term "hydrocarbon moiety" as used herein refers to an
alkyl group of up to 20 carbon atoms (i.e., alkanes, alkenes or
alkynes) that may be saturated, unsaturated, branched, linear, or
cyclic; or an aralkyl group of up to 20 carbon atoms in which the
alkyl portion may be saturated, unsaturated, branched, linear or
cyclic; or an aryl group of up to 20 carbon atoms. Suitably, the
hydrocarbon moiety has from 2 to 15 carbon atoms, or from 5 to 10
carbon atoms. A "substituted hydrocarbon moiety" refers to a
hydrocarbon moiety, as defined herein, in which at least one carbon
atom is substituted with an oxygen, a sulfur, or a nitrogen atom.
The substituent may be, for example, oxo, alkoxy, alkoxycarbonyl,
hydroxy, esters, thioethers, amino, alkylamine, or carbamoyl.
[0058] Examples of suitable aminosilane compounds useful in the
practice of the present invention include, but are not limited, to
aminopropylsilane, propylethylenediaminesilane,
N-[3-(trimethoxysilyl)propyl]ethylenediamine, and
N-[3-(trimethoxysilyl)propyl]diethylenetriamine.
[0059] An NTA-modified solid support, as described herein, may be
produced by contacting a solid support having a free --NH.sub.2
moiety to form an amide bond between nitrilotriaceticacid and the
amine group of the support. Nitrilotriacetic acid acts as a
chelating agent capable of forming stable complexes with polyvalent
metal ions.
[0060] Any solid support is acceptable for use in the production of
an NTA-modified solid support, provided that it has an amine moiety
that can be modified, or that the solid support can be made to
contain a modifiable amine group. Suitable solid supports for use
in the manufacture of NTA-modified solid supports have a plurality
of free NH.sub.2 moieties. One skilled in the art would be able to
attach a free amine functionality to a solid support by chemically
modifying the surface of the solid support. See, e.g., Greg T.
Hermason, A. Krishna Mallia, Paul K. Smith, Immobilized Affinity
Legand Techniques, Academic Press (1992). In addition, suitable
solid supports with free NH.sub.2 moieties capable of binding to
the NTA to form an NTA-modified solid support according to the
present invention are commercially available. These include, but
are not limited to, agarose-based supports sold by Sigma-Aldrich
Inc. (St. Louis, Mo.); latex-based supports sold by International
Dynamics Corporation, (Longwood, Fla.); polystyrene-based supports
sold by Bangs Laboratories Inc., (Fishers, Ind.); Spherotech,
(Libertyville, Ill.); and Dynal Biotech, (Lake Success, N.Y.).
[0061] In another aspect, the present invention provides
metal-modified solid supports. The metal-modified solid support, as
described herein, may be produced by contacting the NTA-modified
solid support described above with a metal ion solution to form the
metal-modified solid support. The metal ion solution may be
comprised of metal ion salts, wherein the salts include, but are
not limited to chloride, sulfate, phosphate, acetate, carbonate,
citrate, acetylacetonate, bromide, fluoride, iodide, nitrate and
oxalate salts. The metal concentration may be from less than about
10.sup.-6 M to about 1 M. Typically, the metal ion concentration in
solution may be in the range of about 0.1 M to about 1 M. It is
envisioned that the metal ion solution may be composed of only one
metal ion or a mixture of different metals. Suitably, a
tetradentate complex may be formed between the metal ion and the
NTA-modified solid support. See, e.g., New multidentate ligands.
XV. Chelating tendencies of diglycine-N,N-diacetic acid,
triglycine-N,N-diacetic acid, and tetraglycine-N,N-diacetic acid,
Inorganic Chemistry (1974), 13(3), 550-9.
[0062] By a "metal ion" as it is used in the context of a
metal-modified solid support, it is meant any metal with a
oxidation state between +1 and +6. Suitably, the metal may be
nickel, copper, cobalt, iron, zinc, or gallium. Additionally the
following metal ions are considered suitable for the present
invention: iron (III), copper (II), cobalt (II), nickel (II), zinc
(II), cerium (III), magnesium (II), calcium (II), galium (III),
chromium (III), indium (III), lanthanum (III), lutetium (III),
scandium (III), thallium (III), ytterbium (III), thorium (IV),
uranate (II) silver (I), gold (I) and copper (I). One skilled in
the art would be able to select a suitable metal depending on the
material to be separated. Also, it is envisioned that the bound
metal ions may be stripped from the metal-modified support with a
chelating agent, such as ethylene diamine tetraacetic acid (EDTA),
therefore allowing the regeneration of the NTA-modified solid
supports.
[0063] The modified solid supports of the invention are useful in a
number of methods, including, but not limited to, those described
in the Examples or in other sections of the specification. As one
skilled in the art will appreciate, the supports of the invention
may be supplied or used in a variety of different forms, depending
on particular requirements of the application. For example, the
modified solid supports may be used in a column, a spin column, in
wells of a microtiter plate, incorporated into or formed as a
filter, as a device implantable in a mammal, or disposed in a
transfer means (e.g., a pipet tip).
[0064] Starting material used in the methods of the invention may
include any material comprising, or suspected of comprising, target
material, and may optionally comprise non-target material. Starting
material includes material taken directly from a biological source
(e.g., a cell culture, spent culture medium, or a cell lysate) or
an environmental source (e.g., water or air) as well as material
that has been processed or partially purified. Starting material
may include the target or non-target material in any form (e.g., in
solution or in suspension). The starting material may be derived
from eukaryotic or prokaryotic sources, including cultured
eukaryotic or prokaryotic cells, and may include either recombinant
or naturally occurring biomolecules. The starting material may
include, for example, a crude cell lysate, including lysates used
in expressions systems, including, but not limited to, cell-free
lysates such as E. coli S-30, wheat germ, and rabbit reticulocyte
lysate. The starting material may be spent bacterial or cell
culture medium into which target materials were secreted. A
suitable starting material may include a complex mixture of
proteins. The starting material may include bacterial, yeast,
fungal, or viral material, plant or animal material, including
products thereof (e.g., whole blood, plasma, serum, milk, and the
like). The starting material may also include fluids such as water,
air, or urine.
[0065] In the methods of the invention, a starting material is
contacted with a modified solid support of the invention to form a
complex between a target material and the support. For the sake of
simplicity, the material recovered by mechanical separation of the
contacted starting material from the particles will be referred to
as "flow through", regardless of the means by which separation was
effected.
[0066] The methods of the present invention depend on the ability
of target or non-target material in starting material to complex
with a composition of the invention to achieve a particular effect,
i.e., a change in the spatial relationship between the target
material and the starting material, or a component thereof. This
effect may be described, with reference to a target or non-target
material, as removing, separating, isolating, purifying,
fractionating, or the like. These terms are not intended to limit
the invention. One of skill in the art will appreciate that the
terms are relative, rather than absolute, and may be used
interchangably.
[0067] In some applications, the method of the invention is
performed in order to effect removal of an undesired target
material from a starting material (e.g., removal of potentially
toxic metal ions from a fluid such as water). In other cases, the
object is to isolate or purify a particular target material from a
complex mixture comprising the target material and non-target
material, or to fractionate target and non-target material.
[0068] Target material such as polypeptides isolated or purified by
the method of the present invention are suitable for use in many
downstream applications. Further isolation, characterization, or
quantitation of isolated target material may be performed by any of
a variety of techniques, including, but are not limited to,
two-dimensional gel electrophoresis, mass spectrometry, X-ray
diffraction, nuclear magnetic resonance, protein chips (array-based
or matrix-based), and yeast two-hybrid system.
[0069] The term polypeptide as used herein includes a polymer of
three or more amino acid units linked via peptide bonds, and may
include proteins. Polypeptides may include a single chain, or two
or more homologous or heterologous polypeptide chains, as in the
case of native proteins have multiple subunits, or in the case of
diverse polypeptides that interact or complex with each other
(e.g., antibody-antigen complexes, or enzymes that have a protein
as a substrate). Polypeptides may include either denatured proteins
or native proteins. Suitable polypeptides may include metal binding
moieties or surface-active amino acids that can act as electron
density donors or acceptors (e.g., lysine, arginine, histidine,
cysteine, glutamic acid, or aspartic acid). A polypeptide having
greater than three histidine or cysteine residues on its surfaces
is particularly well suited for purification according to the
methods of the instant invention. The histidines or cysteines may
be naturally-occurring histidines (e.g., as found in hemoglobin,
myoglobin, and other heme-containing proteins), or may be added to
the polypeptide through genetic engineering techniques known to one
skilled in the art.
[0070] Suitable polypeptides include, without limitation, metallo
proteins, hormones, receptors, enzymes, storage polypeptides, blood
polypeptides, antibodies, membrane polypeptides, phosphorylated
polypeptides, cytoplasmic polypeptides, secretory polypeptides,
organelle polypeptides, polypeptide-nucleic acid complexes,
multi-protein complexes, mutant polypeptides produced by genetic
engineering techniques known to one skilled in the art.
[0071] Target material, including target polypeptides, may be
modified or designed to include an "affinity tag" to facilitate
separation of the target material from non-target material lacking
the affinity tag. The affinity-tagged target polypeptide may be
formed by chemical or recombinant DNA methods known in the art.
Suitably, the affinity tags may be added to the N- or C-terminus,
or to both the N- and C-termini, by genetically engineering a
polynucleotide sequence encoding the target polypeptide to include
the affinity tag. Sequences that encode suitable affinity tags may
also be engineered such that affinity tag is at an internal site on
the target polypeptide. Suitable affinity tags may include, for
example, histidine (His) tags (e.g., polyhistidine tails) and metal
binding domains. Other affinity tags suitable for use in the
present invention may include poly-arginine tag, Strep-tag,
calmodulin binding peptide, maltose binding protein,
glutathione-5-transferase (GST), ubiquitine, or biotin/avidin.
[0072] The Examples below describe separation of specific
his-tagged proteins (e.g., His-luciferase, His-RNaseHI, and
His-methionyl tRNA transferase). The methods of the invention were
found to be effective in isolating other his-tagged proteins
(His-endostatin, His-Tau, His-Karyopherin-alpha 2, His-ubiquitin,
His-osteopontin, and His-calcinuerin B alpha proteins) (data not
shown). One skilled in the art will appreciate that the methods may
be used to purify any his-tagged protein.
[0073] It is envisioned that the methods of the invention may be
used to isolate any target molecule of interest from non-target
molecules in a starting material, provided that the target molecule
has a differential tendency to form a complex with the compositions
of the invention, relative to at least one species of non-target
molecule present in the starting material.
[0074] An NTA-modified solid support may be used to remove toxic
metals from water, air, blood, or other fluids of interest. For
example, the NTA-modified solid may be used to remove and/or
recover potentially harmful or toxic metals, such as aluminum,
arsenic, bismuth, antimony, excess calcium, excess iron, gold,
zinc, magnesium, mercury, cadmium, lead, copper and silver, from
industrial waste waters and or from water destined for human
consumption. of particular concern are lead salts that can leach
from the pipes and solder joints of home plumbing. The NTA-modified
solid supports may be used in a manner similar to chelating agents
to remove heavy metal ions from water (e.g., U.S. Pat. No.
4,500,494), to analyze heavy metal ions in water using chelating
compounds in conjunction with a filter to trap the metal ions
(e.g., U.S. Pat. No. 4,080,171), to purify water (e.g., U.S. Pat.
No. 4,348,328), or, in conjunction with a resin, to recover heavy
metal ions from liquid (e.g., U.S. Pat. No. 4,220,726).
[0075] The NTA-modified solid supports of the present invention may
also be useful in a number of other applications in which it is
desirable to extract, deactivate or remove metals from fluids,
e.g., removing calcium from plasma to convert the plasma to serum,
or wiping up spills of radioactive metallic ions in laboratories.
The NTA-modified supports may be employed to remove toxic metals
from individuals with lead or mercury poisoning.
[0076] Interference with or depletion of certain metal ions has
been reported as having a role in health conditions. Accordingly,
the NTA-modified solid supports of the present invention may be
used as a diagnostic tool for detecting and extracting
metal-associating molecules indicative of the disease state or
predisposition to a disease.
[0077] The NTA-modified solid supports may be used to prepare
chelating immunostimulating complexes in a manner similar to the
general approaches described, for example, in U.S. Pat. No.
6,428,807.
[0078] Metal-modified solid supports may be used to separate target
material (e.g., polypeptides or nucleic acids) from non-target
material in a starting material. For example, the metal-modified
support may be used to separate his-tagged polypeptides from other
molecules present in a starting material. The starting material may
optionally be adjusted to include imidazole in a concentration of
from about 0 to about 60 mM. The his-tagged proteins can be eluted
from the support using any suitable buffer. A suitable buffer could
contain imidazole in a concentration of from about 60 mM to about 1
M. Other suitable elution buffers are those having a pH lower than
the isoelectric point (pI) of the protein of interest, suitably
less than about pH 5. Other suitable elution buffers include
buffers with competing chelating agents such as EDTA or EGTA,
trifluoroacetic acid, L-histidine, dipeptides, histidine peptides
or polymers, or imidazole-like polymers.
[0079] The metal-modified solid support may be used alone or in
conjunction with other purification methods, including, for
example, methods using an amine-modified solid support.
[0080] The metal-modified solid support may also be used to remove
endotoxins from a starting material. Suitably, the term endotoxin
refers to the lipopolysaccharide complex associated with the outer
membrane of certain species of gram-negative bacteria such as E.
coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, or
any other endotoxin-producing pathogenic bacterium.
[0081] The metal-modified solid support may be used to isolate or
identify low-abundance proteins, membrane proteins, or
phosphorylated proteins.
[0082] The metal-modified support may be used to separate nucleic
acids, as described for other IMAC resins in WO 02/42398A2.
[0083] As described in the Examples below, the metal-modified solid
support of the present invention as well as other solid supports
comprising an immobilized metal chelating agent (e.g., nickel
agarose beads commercially available from Qiagen) were found to
allow detection of proteins complexed to the solid support using a
detectable label. In the Examples, Coomassie, fluorescein, or
Bodipy were found to complex with proteins immobilized to the solid
supports in a quantitative fashion. It is envisioned that any
suitable dye or fluorescent label may be used to detect proteins
complexed with a solid support comprising an immobilized metal
chelating agent. Other examples of suitable detectable labels
include, without limitation, remazol brilliant blue R, eosin
isothiocyanate, reactive orange, procion red, eosin iodoacetamide,
reactive black 5, reactive orange 14, malachite green
isothiocyanate, rhodamine isothiocyanate, remasol brilliant violet
5R, rhodamine, and coumarin.
[0084] The metal-modified solid support may also be used to isolate
or evaluate polypeptide-polypeptide complexes or interactions;
screen for polypeptide function; isolating antibodies, antigens, or
antibody-antigen complexes; quantitating affinity-tagged
polypeptides; diagnostic screening for diseases; antibody
screening; antagonist and agonist screening for drugs; reporter
gene assays; producing polypeptide expression libraries, producing
polypeptide libraries from cells; producing polypeptide
microarrays; screening genetically engineered enzymes; co-isolating
interacting molecules (e.g., co-factors); reducing in vivo
concentrations of an endotoxin; tissue profiling; or cell
profiling.
[0085] As detailed in the Examples below, amine-modified solid
supports were found to be useful in a variety of applications.
Amine-modified solid supports permit facile separation of
hemoglobin from other materials present in a starting material,
isolation of membrane vesicles, purification of membrane proteins,
and concentration or purification of cells.
[0086] An amine-modified solid support may be used to remove
hemoglobin from a starting material. As demonstrated in the
Examples below, hemoglobin does not bind to amine-modified silica
magnetic particles. Separation of hemoglobin from other proteins
may useful in any application in which hemoglobin is present. For
example, purification of proteins expressed in a reticulocyte
lysate expression system can be enhanced by removing hemoglobin by
contacting the lysate with an amine-modified solid support. Removal
of hemoglobin is particularly useful in applications employing
fluorescent-based detection, because hemoglobin interferes with
detection of fluorescently labeled proteins by reducing the signal
to noise ratio. In addition to facilitating removal of hemoglobin,
the method of the invention may be expected to allow removal of
other proteins containing heme groups, such as myoglobin.
[0087] An amine-modified solid support may be used to isolate
membrane-associated proteins for subsequent identification or
characterization. In the examples below, membrane proteins or
membrane-associated proteins were expressed in cell-free lysate in
presence of microsomal membrane vesicles and separated from other
materials present in the lysate by contacting the lysate with an
amine-modified solid support such that the membranes formed a
complex with the support. This method can be adapted for use in
screening collections of in vitro expressed proteins to facilitate
isolation and identification of membrane proteins and is suitable
for use in high throughput screening of such proteins. As can be
seen from the Examples, the method may optionally employ a
non-ionic detergent, which facilitates recovery of membrane
proteins.
[0088] As described in the Examples, an amine-modified solid
support may be used in conjunction with other purification methods,
such as metal-modified solid supports, to purify molecules of
interest, including affinity-tagged polypeptides under denaturing
or non-denaturing conditions. The order in which purification steps
using amine-modified solid support are performed relative to other
steps in a purification scheme may be altered, depending on the
nature of the target material and any non-target material that may
be present.
[0089] It is envisioned that the specific target proteins bound to
the amine-modified solid support may be subsequently eluted under
appropriate conditions specific to the particular target protein.
Suitable fractionation conditions are known to skilled researchers
in the art. As shown in the examples, target proteins containing
histidine residues, such as a his-tag, do not bind to
amine-modified solid supports. It is envisioned that the
amine-modified solid supports of the present invention may
facilitate separation of target proteins that have been genetically
engineered to include moieties that reduce binding to
amine-modified solid supports, including, but not limited to, metal
binding moieties or surface-active amino acids. Depending on the
technique used to separate his-tagged target polypeptides, unbound
his-tagged polypeptide may appear in the flow-through. The unbound
his-tagged polypeptide may be further purified using a
metal-modified solid support.
[0090] The following non-limiting Examples are intended to be
purely illustrative.
EXAMPLES
Example 1
Preparation of Metal-Modified
3-[[[Bis(carboxymethyl)amino]acetyl]-amino]propyl Silica Magnetic
Particles
a) Preparation of 3-Aminopropyl-Modified Magnetic Silica
Particles
[0091] 3-Aminopropyl-modified magnetic silica particles were
prepared as follows. A 50-ml aliquot of
3-aminopropyltrimethoxysilane was added to a stirred solution of
methanol (900 mL) followed by addition of water (50 mL). The
mixture was added to 100 g of magnetic silica particles (MP-50,
W.R. Grace, Columbia, Md.). The particles were kept in suspension
for 4 hr at room temperature using intermittent agitation. The
residual methanol/silane/water solution was removed and the support
particles were washed with 3.times.1.2 L of water then resuspended
in 1 L of methanol. The 3-aminopropyl-modified magnetic silica
particles were collected by filtration and dried under vacuum.
Elemental analysis confirmed the composition of the
3-aminopropyl-modified magnetic silica particles: C, 0.75; H, 0.64;
N, 0.30.
b) Preparation of 3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl
Magnetic Silica Particles
[0092] 3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic
silica particles were made by first suspending
3-aminopropyl-modified magnetic silica particles (100 g), prepared
as described above, in N,N-dimethylacetamide (600 mL), adding
triethylamine (31 ml, 210 mmoles), and mixing thoroughly. 200
mmoles of 2,6-diketo-N-carboxymethyl-morpholine (prepared according
to U.S. Pat. No. 3,621,018) in 400 ml of N,N-dimethylacetamide was
added and the resulting mixture was kept in suspension for 4 hr at
room temperature. The unreacted
N,N-dimethylacetamide/anhydride/triethylamine solution was removed
and the particles were washed with 3.times.1.2 L of water.
Elemental analysis confirmed the composition of
3-[[[bis(carboxymethyl)amino]acetyl]amino]-propyl-modified magnetic
silica particles: C, 1.06; H, 0.61; N, 0.17.
c) Preparation of Nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]propyl Magnetic Silica
Particles
[0093] Nickel (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were prepared by suspending 100 grams of
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, prepared as described above, in a 250 mM nickel (II)
chloride solution (1 L) for 4 hours at room temperature. The excess
nickel solution was removed and the resulting solid support was
washed with five times with water.
[0094] Modified particles similar to those described above in
Example 1(a)-(c) were prepared using starting particles other than
magnetic silica particles from W.R. Grace. Other silica gels that
have been used in steps (a)-(c) were supplied by: Sigma-Aldrich
Corp (St. Louis, Mo.) (23,681-0, 23,682-9, and 23, 684-5); Silicyle
Inc. (Quebec, CA) (S10030M, 10040M, 100300T, S10040T, and R10030M);
or J. T. Baker (Philipsburg, N.J.) (7314-02 and 7315-20). The
commercial silica gels contained particles having diameters in the
range of about 5 to about 500 microns, and pore sizes in the range
of about 40 to about 1000 Angstroms.
d) Preparation of Cobalt (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]propyl Magnetic Silica
Particles
[0095] Cobalt (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were prepared by suspending 100 mg of a
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, prepared as described above, in a 250 mM cobalt (II)
chloride solution for two minutes at room temperature. The excess
cobalt solution was removed and the resulting magnetic silica
particles were washed 5 times with water.
e) Preparation of Copper (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]propyl Magnetic Silica
Particles
[0096] Copper (II)
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were prepared by suspending 100 mg of a
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, prepared as described above, in a CuCl.sub.2 (250 mM)
solution for two minutes at room temperature. The copper solution
was removed and the resulting magnetic silica particles were washed
three times with water.
f) Preparation of Zinc (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]propyl Magnetic Silica
Particles
[0097] Zinc (II) 3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl
magnetic silica particles were prepared by suspending 100 mg of a
3-[[[bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, prepared as described above, in a ZnCl.sub.2 (250 mM)
solution for two minutes at room temperature. The zinc solution was
removed and the resulting magnetic silica particles were washed
three times with water.
Example 2
Preparation of Metal-Modified
3-[[[Bis(carboxymethyl)amino]acetyl]-amino]propyl Silica Gel
(a) Preparation of 3-Aminopropyl-Modified Silica Gel
[0098] 3-Aminopropyltrimethoxysilane (125 mL) was added to a
stirred solution of methanol (2000 mL) followed by addition of
water (125 mL). This mixture was added to 250 g of silica gel
(S10040T, 1000 angstrom, Silicycle, Inc, Quebec, Canada) and the
resulting mixture was kept in suspension for 4 hr at room
temperature. After allowing the resin to settle the residual
methanol/silane/water solution was decanted, the particles were
washed with water (3.times.2.5 L) and resuspended in 2 L of
methanol. The aminosilane-modified solid support was collected by
filtration and dried under vacuum. Elemental analysis confirmed the
composition of aminopropyl-modified solid support: C, 0.46; H,
0.30; N, 0.19.
(b) Preparation of
3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl Silica Gel
[0099] 3-Aminopropyl-modified solid support (100 g) prepared as
described above was suspended in N,N-dimethylacetamide (100 mL) and
triethylamine (31 mL, 210 mmoles) was added to the mixture. This
suspension was mixed thoroughly then 200 mmoles of
2,6-diketo-N-carboxymethylmorpholine (prepared according to U.S.
Pat. No. 3,621,018, the contents of which are incorporated herein
in its entirety) in 400 mL of N,N-dimethylacetamide was added and
the resulting mixture was kept in suspension for 4 hr at room
temperature. The unreacted
N,N-dimethylacetamide/anhydride/triethylamine solution was removed
and the solid support was washed with 4.times.1.2 L of water.
Elemental analysis confirmed the composition of
3-[[[bis(carboxymethyl)amino]acetyl]amino]propyl solid support: C,
0.94; H, 0.32; N, 0.28.
(c) Preparation of Nickel (II)
3-[[[Bis(carboxymethyl)amino]acetyl]-amino]propyl Silica Gel
[0100] A portion of
3-[[[bis(carboxymethyl)amino]acetyl]amino]propyl solid support
prepared as described above was suspended in 250 mM nickel (II)
chloride solution for 4 hr at room temperature. The excess nickel
solution was removed and the resulting solid support was washed 5
times with water.
Example 3
Preparation of Propylethylenediamine-Modified Magnetic Silica
Particles
[0101] N-[3-(Trimethoxysilyl)propyl]ethylenediamine (2 mL) was
added to a stirred solution of magnetic silica particles (2 g) in
95% methanol (8 mL). The resulting mixture was kept in suspension
for 4 hr at room temperature. The residual methanol/silica solution
was removed and the particles were washed with methanol (5.times.40
mL) and dried under vacuum. Elemental analysis confirmed the
composition of aminopropylethylenediamine-modified silica magnetic
solid support: C, 0.97; H, 0.70; N, 0.45.
Example 4
Preparation of Propylethylenediamine-Modified Silica Gel
[0102] N-[3-(Trimethoxysilyl)propyl]ethylenediamine (2 mL) was
added to a stirred solution of silica particles (1.0 g of Davisil,
grade 644 silica gel, 100-200 mesh, 150 A pore size) in 95%
methanol (8 mL). The resulting mixture was kept in suspension for 4
hr at room temperature. The residual methanol/silica solution was
removed and the particles were washed with methanol, 5.times.40 mL,
and dried under vacuum. Elemental analysis confirmed the
composition of aminopropylethylenediamine-modified silica solid
support: C, 5.82; H, 1.49; N, 2.44.
[0103] The foregoing description of the invention is exemplary for
purposes of illustration and explanation. It will be apparent to
those skilled in the art that changes and modifications are
possible without departing from the spirit and scope of the
invention. It is intended that the following claims be interpreted
to embrace all such changes and modifications.
Example 5
Removal of Hemoglobin and Fractionation of Target Proteins Using
3-Aminopropyl-Modified Magnetic Silica Particles
[0104] The ability of 3-aminopropyl modified magnetic silica
particles to fractionate proteins and to remove hemoglobin from
rabbit reticulocyte lysate spiked with .beta.-galactosidase and
calf intestinal alkaline phosphatase was evaluated in parallel with
Q-sepharose (BioRad, Foster City, Calif.) and DEAE sepharose
(Sigma-Aldrich, Milwaukee, Wis.). A sample containing untreated
rabbit reticulocyte lysate (100 .mu.l), 1 ml 20 mM Tris-buffer (pH
8.3), 8 .mu.l stock .beta.-galactosidase (Promega Corp.), 40 .mu.l
calf intestinal alkaline phosphatase (Promega Corp.) was prepared.
Aliquots (400 .mu.l) of the sample were to applied to
pre-equilibrated 3-aminopropyl-modified magnetic silica particles
(40 mg), prepared as described above, in 1.5 ml Eppendorf tubes,
and mixed at room temperature. The particles were separated from
the supernatant (or flow through) by placing the tubes on a
magnetic stand, and the supernatant was removed and reserved. The
particles were washed twice with 400 .mu.l 200 mM Tris buffer (pH
8.3). Bound proteins (.beta.-galactosidase and calf intestinal
alkaline phosphatase) were eluted with a sequential application of
20 mM Pipes buffer (pH 6.7), 20 mM sodium citrate (pH 5.0),
followed by two applications of 1 M ammonium acetate in 20 mM
Tris-buffer (pH 8.3). The separation procedure as described above
for the 3-aminopropyl-modified magnetic silica particles was used
to evaluate hemoglobin removal and protein fractionation on
Q-sepharose or DEAE sepharose.
[0105] The hemoglobin content of each supernatant was measured by
diluting an aliquot of the supernatant 1:20 with water and
measuring the absorbance at 415 nm.
[0106] .beta.-galactosidase activity was measured as follows.
Promega 2.times. Assay Buffer [E203A, 14041201] was diluted 1:1
with nanopure water, and 490 .mu.l buffer was added to 1.5 ml
plastic microfuge tubes. Ten microliters of test fractions was
added to the tubes. As a control, 10 microliters of 20 mM Tris
buffer pH 8.3 was added in place of the test fraction. The tubes
were incubated at RT for 45 min. A 0.5 ml aliquot of sodium
carbonate solution [Promega E202A, 14679601] was added to each
tube, and the absorbances of the solutions were read at 420 nm,
using the control tube as a blank.
[0107] Phosphatase activity was measured as follows. A phosphatase
assay reagent of a saturated solution of p-nitrophenyl phosphate
was prepared by mixing 48 ml of 100 mM Tris buffer pH 8.3 with
p-nitrophenyl phosphate solution, which was made by suspending
solid p-NPP (Sigma Chemical Co.) in 100% ethanol in an amount that
would generate a 20 mM solution if all the material would dissolve.
Nine hundred microliters of the saturated solution was placed in
1.5 ml microfuge tubes and 2 .mu.l of the fractions added to the
tubes. As a control, 2 ul of 20 mM Tris pH 8.3 was added in place
of the test fractions. The tubes were incubated 120 min at
37.degree. C., and the absorbance at 420 nm was measured after
blanking the spectrophotometer with the control solution.
[0108] The results are presented graphically in FIG. 1A
(aminopropyl-modified magnetic silica particles), FIG. 1B
(Q-sepharose), and FIG. 1C (DEAE sepharose). When
aminopropyl-modified magnetic silica particles were used to
fractionate proteins in the starting material, most of the
hemoglobin was found in the unbound flow through fraction (FIG.
1A). In contrast, with both Q-sepharose and DEAE sepharose, the
largest percentage of hemoglobin bound to the resin and was eluted
by the 20 mM Pipes buffer (pH 6.7), and a substantial amount of
hemoglobin eluted by 20 mM sodium citrate (pH 5.0). Fractionation
of .beta.-galactosidase and calf intestinal alkaline phosphatase
was achieved by eluting bound protein from aminopropyl-modified
magnetic silica particles using different elution buffers (FIG.
1A).
Example 6
Separation of FluoroTect-Labeled Luciferase from Hemoglobin Using
3-Aminopropyl Silica Magnetic Particles
[0109] 3-aminopropyl-modified magnetic silica particles were used
to fractionate proteins from rabbit reticulocyte lysate. FluoroTect
Green in vitro translation labeling system (Promega Corp., Madison,
Wis.) was used to express FluoroTect-labeled luciferase in rabbit
reticulocyte lysate according to the manufacturer's instructions.
Following translation, 20 .mu.L of the translation reaction mixture
was mixed with 5 mg aminopropyl-modified silica magnetic particles
(100 mg/ml) pre-equilibrated in 20 mM MOPS buffer (pH 6.8) in 1.5
ml Eppendorf tubes. The particles were resuspended by mixing at
room temperature. The particles were separated from the supernatant
by placing the tubes on a magnetic stand and removing the
supernatant, which was reserved for subsequent analysis. The
particles were washed twice with 1 ml 20 mM MOPS buffer (pH 6.8).
The particles were treated with either 2 M ammonium acetate (pH
6.5) or 1 M NaCl, and the purification fractions were collected and
analyzed by SDS-PAGE (FIG. 2). With reference to FIG. 2, the gel
was loaded as follows: Lane M, protein molecular weight marker
(Promega Corp.); lanes 1 and 2, control containing 101 of reaction
mixture+40 .mu.l of buffer; lanes 4 and 5, ammonium acetate (pH
6.5) eluate; lanes 6-9, 1 M NaCl eluate.
[0110] As can be seen by comparing lanes 1 and 2 with lanes 4 and 5
of the gel shown in FIG. 2, luciferase was recovered in the
fraction eluted with ammonium acetate (pH 6.5), with a significant
reduction in hemoglobin, indicating that a substantial amount of
hemoglobin appeared in the flow through fraction and did not bind
to the particles.
Example 7
Capturing Membrane Vesicles Used in In Vitro Translation Using
3-Aminopropyl Magnetic Silica Particles
[0111] In vitro protein synthesis: Core glycosylation control mRNA
(S. cerevisiae alpha factor) was translated using rabbit
reticulocyte lysate (Promega Corp. Madison, Wis.) for 60 min at
30.degree. C. Reaction mixtures (25 .mu.l), prepared as summarized
in Table 1, contained 17.5 .mu.l of reticulocyte lysate, 0.5 .mu.l
of RNasin (40 units/.mu.l), 0.5 .mu.l of 1 mM amino acids (minus
methionine), 20 .mu.Ci of L-[.sup.35S]methionine (Amersham), 2
.mu.l of 0.1 .mu.g/.mu.l core glycosylation control RNA, and,
optionally, either canine microsomal membrane (Promega Corp.) or
HeLa microsomal membrane vesicles (prepared at Promega Corp.) from
HeLa cell line (HeLa-S3) (Biovest International Inc., Englewood
Cliffs, N.J.). Reactions were analyzed by SDS-PAGE on a 4-20% Novex
gel, transferred to a sheet of PVDF, and exposed to a
PhosphorImager cassette for 16 hr.
TABLE-US-00001 TABLE 1 1 2 3 4 5 6 7 8 9 10 Rabbit 17.5 17.5 17.5
17.5 17.5 17.5 17.5 17.5 17.5 17.5 RetiLysate Water 2.5 1.5 1.5 3.5
3.5 3.5 2.5 2.5 3.5 3.5 Rnasin 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.5 Amino Acid 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 (-Met) RNA
-- 1.0 1.0 1.0 1.0 -- 1.0 1.0 1.0 1.0 template* 35 S Met 2.0 2.0
2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 CMM** 2.0 2.0 2.0 -- -- HMM*** 1.0
1.0 1.0 -- -- Particles + + - + - + + - + - *Glycosylation control
mRNA (S. cerevisiae .alpha.-factor) **Canine Microsomal membrane
***HeLa Microsomal membrane
[0112] Aliquots of 3-aminopropyl magnetic silica particles (60
mg/ml) were added to 25 .mu.l of the translation reaction mixture,
as indicated in Table 1. SDS buffer 4.times. (10 .mu.l) was added
to .about.20-25 .mu.l of the supernatant (flow through), heat
denatured and examined by SDS-PAGE. The particles were washed once
with 1 ml 20 mM MOPS buffer (pH 6.8) and proteins eluted with 25
.mu.l of 1 M NaCl in MOPS buffer (pH 6.8). The eluate was mixed
with 10 .mu.l of 4.times.SDS buffer, heat denatured, and examined
by SDS-PAGE. 10 .mu.l of 1 M NaCl in MOPS buffer (pH 6.8) and 10
.mu.l of 4.times.SDS buffer were added to the aminopropyl magnetic
silica particles, heat denatured and examined by SDS-PAGE. As a
control, aliquots (3 .mu.l) of reaction mixtures not contacted with
aminopropyl magnetic silica particles were treated with 20 .mu.l of
20 mM MOPS (pH 6.8) and 10 .mu.l of 4.times.SDS buffer, heat
denatured and examined by SDS-PAGE. (FIG. 3). The results of this
experiment demonstrate that proteins expressed in vitro and
modified post-translationally can be separated and analyzed using
3-aminopropyl magnetic silica particles.
Example 8
Identifying and Purifying Membrane Proteins
[0113] S. cerevisiae factor mRNA was translated using rabbit
reticulocyte lysate (Promega corporation, Madison, Wis.) in a 50
.mu.l reaction for 60 minutes in the presence of HeLa microsomal
membrane vesicles prepared at Promega Corporation from HeLa cell
line (HeLa-S3) (Biovest International Inc.). The translation
reaction (50 .mu.l each) contained 35.0 .mu.l of reticulocyte
lysate, 1.0 .mu.l of RNasin (40 units/.mu.l), 1.0 .mu.l of 1 mM
amino acids (minus methionine), 20 .mu.Ci of L-[.sup.35S]methionine
[Amersham Biosciences, Sunnyvale, Calif.], 2 .mu.l of 0.1
.mu.g/.mu.l core glycosylation control RNA, and HeLa microsomal
membrane vesicles. A portion of the reaction mixture was treated
with 4.times.SDS buffer, heat denatured, and analyzed by SDS-PAGE
on a 4-20% Novex gel. The gel was transferred onto a sheet of PVDF
and exposed to a PhosphorImager cassette for 16 hr for analysis
(FIG. 4).
[0114] Below is a summary of the alternative treatments the various
reaction mixtures were subjected to prior to separation by
SDS-PAGE.
[0115] (1) After the translation reaction, the reaction mixture was
added to the 3-aminopropyl magnetic silica particles either in a
1:1 ratio (i.e., 10 .mu.l of mixture to 10 .mu.l of particles) or
in a 1:.about.1.7 ratio (i.e., 30 .mu.l of mixture to 50 .mu.l of
particles). The particle mixture was washed 1.times. with 1 ml of
20 mM MOPS buffer. The particles were treated with 20 .mu.l of 1 N
NaCl in 20 mM MOPS buffer and 10 .mu.l of 4.times.SDS buffer. The
particle mixture was heat denatured and the supernatant was
analyzed by SDS-PAGE, as shown in FIG. 4, lanes 1A, 1B and 2A,
2B.
[0116] (2) After the translation reaction, 3-aminopropyl magnetic
silica particle capture was followed by limited proteolysis using
proteinase K. The reaction mixture was added to the 3-aminopropyl
magnetic silica particles either in a 1:1 ratio (i.e., 10 .mu.l of
mixture to 10 .mu.l of particles) or in a 1:.about.1.2 ratio (i.e.,
40 .mu.l of mixture to 50 .mu.l of particles). The particles were
washed 1.times. with 1 ml of 20 mM MOPS buffer and treated with 10
.mu.l of 50 mM Tris/CaCl.sub.2 buffer and 2 .mu.l of 50 mM
CaCl.sub.2. The mixture was incubated on ice for 10 min. Following
incubation, proteinase K (1 .mu.l of 1 mg/ml) or, alternatively,
proteinase K (1 .mu.l of 1 mg/ml) and Triton X-100 (1 .mu.l of 10%)
was added and the resulting mixture was incubated on ice for 1 hr.
The proteinase K reaction was stopped by adding 2 .mu.l of 2 mg/ml
PMSF. Also added was 5 .mu.l of 20 mM MOPS buffer and 10 .mu.l of
4.times.SDS. The reaction mixture was heat denatured and analyzed
by SDS-PAGE. When 40 .mu.l of mixture was used in the reaction, the
following reagents were also increased by 4-fold: Tris/CaCl.sub.2
buffer, 50 mM CaCl.sub.2, 1 mg/mL Proteinase K, Triton X-100 (1
.mu.l of 10%), PMSF 2 mg/mL. A small amount of reaction mixture was
used for gel analysis. Then, to the reaction particles was added 10
.mu.l of 4.times.SDS buffer. The particle mixture was heat
denatured and the supernatant was analyzed by SDS-PAGE, as shown in
FIG. 4 lanes 3A, 3B and 4A, 4B or as shown in FIG. 4 lanes 7A, 7B
and 8A, 8B using Triton X-100 to solubilize membrane proteins.
[0117] (3) After the translation reaction, proteinase K treatment
followed by 3-aminopropyl magnetic silica particle capture of the
membrane vesicles were performed. The reaction mixture (i.e., 10
.mu.l) was added to 2 .mu.l of 50 mM CaCl.sub.2. The mixture was
incubated on ice for 10 min. Proteinase K (1 .mu.l of 1 mg/ml) or
Proteinase K (1 .mu.l of 1 mg/ml) and Triton X-100 (1 .mu.l of 10%)
was added to the reaction mixture and allowed to incubate for 1 hr
on ice. The reaction was stopped by adding 2 .mu.l of 2 mg/ml PMSF.
The 3-aminopropyl magnetic silica particles (10 .mu.l) were added
to the mixture. The particle suspension was allowed to settle and
the particles were washed 1.times. with 1 ml of 20 mM MOPS. To
elute the membrane bound proteins, 20 .mu.l of 1 N NaCl in 20 mM
MOPS buffer was added to the particles. In preparation for SDS-PAGE
analysis, 10 .mu.l of 4.times.SDS buffer was added to the mixture.
The mixture was subsequently heat-denatured and analyzed by
SDS-PAGE. When 40 .mu.l of mixture was used in the reaction, the
following reagents were also increased by 4.times. the volume:
Tris/CaCl.sub.2 buffer, 50 mM CaCl.sub.2, 1 mg/mL Proteinase K,
Triton X-100 (1 .mu.l of 10%), PMSF 2 mg/mL. A small amount of
reaction mixture was used for gel analysis. The reaction particles
were combined with 10 .mu.l of 4.times.SDS buffer, the particle
mixture was heat denatured, and the supernatant was analyzed by
SDS-PAGE, as shown in FIG. 4 lanes 5A, 5B and 6A, 6B or as shown in
FIG. 4 lanes 9A, 9B and 10A, 10B, using Triton X-100 to solubilize
membrane proteins.
[0118] The gel shown in FIG. 4 was loaded as follows: [0119] Lane
M; Marker [0120] Lane 1: RRL+mRNA [0121] Lane 1A:
RRL+mRNA--3-aminopropyl magnetic silica particle capture (10 .mu.l
of reaction mixture+10 .mu.l of 3-aminopropyl magnetic silica
particles) [0122] Lane 1B: RRL+mRNA--3-aminopropyl magnetic silica
particle capture (301 of reaction mixture+50 .mu.l of 3-aminopropyl
magnetic silica particles) [0123] Lane 2: RRL+mRNA+HMM [0124] Lane
2A: RRL+mRNA+HMM--3-aminopropyl magnetic silica particle capture
(10 .mu.l of reaction mixture+10 .mu.l of 3-aminopropyl magnetic
silica particles) [0125] Lane 2B: RRL+mRNA+HMM--3-aminopropyl
magnetic silica particle capture (30 .mu.l of reaction mixture+50
.mu.l of 3-aminopropyl magnetic silica particles) [0126] Lane 3A:
RRL+mRNA--3-aminopropyl magnetic silica particle
capture--Proteinase K treatment (10 .mu.l of reaction mixture+10
.mu.l of 3-aminopropyl magnetic silica particles) [0127] Lane 3B:
RRL+mRNA--3-aminopropyl magnetic silica capture--Proteinase K
treatment (40 .mu.l of reaction mixture+50 .mu.l of 3-aminopropyl
magnetic silica particles) [0128] Lane 4A:
RRL+mRNA+HMM--3-aminopropyl magnetic particle capture--Proteinase K
treatment (10 .mu.l of reaction mixture+10 .mu.l of 3-aminopropyl
magnetic silica particles) [0129] Lane 4B:
RRL+mRNA+HMM--3-aminopropyl magnetic silica particles
capture--Proteinase K treatment (40 .mu.l of reaction mixture+50
.mu.l of 3-aminopropyl magnetic silica particles) [0130] Lane 5A:
RRL+mRNA--Proteinase K treatment--3-aminopropyl magnetic silica
particle capture (10 .mu.l of reaction mixture+10 .mu.l of
amino-silica magnetic particles) [0131] Lane 5B:
RRL+mRNA--Proteinase K treatment--3-aminopropyl magnetic silica
particle capture (40 .mu.l of reaction mixture+50 .mu.l of
3-aminopropyl magnetic silica particles) [0132] Lane 6A:
RRL+mRNA+HMM--Proteinase K treatment--3-aminopropyl magnetic silica
particle capture (10 .mu.l of reaction mixture+10 .mu.l of
3-aminopropyl magnetic silica particles) [0133] Lane 6B:
RRL+mRNA+HMM--Proteinase K treatment--3-aminopropyl magnetic silica
particle capture (40 .mu.l of reaction mixture+50 .mu.l of
3-aminopropyl magnetic silica particles) [0134] Lane 7A:
RRL+mRNA--3-aminopropyl magnetic silica particle
capture--Proteinase K treatment+Triton X-100 (10 .mu.l of reaction
mixture+10 .mu.l of 3-aminopropyl magnetic silica particles) [0135]
Lane 7B: RRL+mRNA--3-aminopropyl magnetic silica particle
capture--Proteinase K treatment+Triton X-100 (40 .mu.l of reaction
mixture+50 .mu.l of 3-aminopropyl magnetic silica particles) [0136]
Lane 8A: RRL+mRNA+HMM--3-aminopropyl magnetic silica particle
capture--Proteinase K treatment+Triton X-100 (10 .mu.l of reaction
mixture+10 .mu.l of 3-aminopropyl magnetic silica particles) [0137]
Lane 8B: RRL+mRNA+HMM--3-aminopropyl magnetic silica particle
capture--Proteinase K treatment+Triton X-100 (40 .mu.l of reaction
mixture+50 .mu.l of 3-aminopropyl magnetic silica particles) [0138]
Lane 9A: RRL+mRNA--Proteinase K treatment+Triton
X-100--3-aminopropyl magnetic silica particle capture (10 .mu.l of
reaction mixture+10 .mu.l of 3-aminopropyl magnetic silica
particles) [0139] Lane 9B: RRL+mRNA--Proteinase K treatment+Triton
X-100--3-aminopropyl magnetic silica particle capture (40 .mu.l of
reaction mixture+50 .mu.l of 3-aminopropyl magnetic silica
particles) [0140] Lane 10A: RRL+mRNA+HMM--Proteinase K
treatment+Triton X-100--3-aminopropyl magnetic silica particle
capture (10 .mu.l of reaction mixture+10 .mu.l of 3-aminopropyl
magnetic silica particles) [0141] Lane 10B:
RRL+mRNA+HMM--Proteinase K treatment+Triton X-100--3-aminopropyl
magnetic silica particle capture (40 .mu.l of reaction mixture+50
.mu.l of 3-aminopropyl magnetic silica particles) [0142] Lane 11:
RRL [0143] RRL: Rabbit reticulocyte lysate [0144] HMM: HeLa
microsomal membrane preparation
[0145] The results of this experiment demonstrate that membrane
proteins expressed in cell free protein expression systems can be
rapidly identified and characterized using 3-aminopropyl magnetic
silica particles.
Example 9
Capture of Bacterial Cells by 3-Aminopropyl Magnetic Silica
Particles
[0146] Bacterial cells (E. coli JM109) were cultured overnight at
37.degree. C. in Luria broth. Culture suspensions (500 .mu.l each)
were transferred to separate Eppendorff tubes. Samples treated with
15% isopropanol were prepared by adding 150 .mu.l of 100%
isopropanol and 350 .mu.l of sterile double distilled water to the
tubes. Samples treated with 30% isopropanol were prepared by mixing
300 .mu.l of 100% isopropanol and 200 .mu.l of sterile double
distilled water with the cells. Samples treated with 15%
isopropanol and 1 M NaCl were prepared by mixing 150 .mu.l of 100%
isopropanol, 200 .mu.l of 5 M NaCl and 150 .mu.l of sterile double
distilled water with the cells. Samples treated with 30%
isopropanol and 1 M NaCl were prepared by mixing 300 .mu.l of 100%
isopropanol and 200 .mu.l of 5 M NaCl with the cells. 3-aminopropyl
magnetic silica particles (10 mg) were added to each tube and mixed
with for 1 minute. No particles were added to the control. Unbound
cells in the supernatant were removed by placing the tubes onto a
magnet to capture the magnetic silica particles and OD.sub.600 of
the supernatant was measured. The results are summarized in FIG. 5,
which shows the OD.sub.600 of unbound cells from: (1) the control
containing no particles; (2) culture treated with double distilled
water; (3) culture treated with 1 M NaCl; (4) culture treated with
15% isopropanol; (5) culture treated with 30% isopropanol; (6)
culture treated with 15% isopropanol and 1M NaCl; and (7) culture
treated with 30% isopropanol and 1M NaCl.
[0147] The 3-aminopropyl magnetic silica particles were able to
bind bacterial cells directly from a culture in one minute or less.
The number of cells bound by the particles was enhanced by the
addition of isopropanol and sodium chloride. The OD.sub.600 of the
supernatant of cultures treated with 15 or 30% isopropanol was
considerably lower than that of untreated cells, which indicates
that 3-aminopropyl magnetic silica particles are effective in
binding cells treated with isopropanol.
Example 10
Separation of Polypeptides Using Nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles
[0148] Preparation of Cell Lysate
[0149] Bacterial cells (E. coli JM109) expressing his-tagged
proteins were grown overnight in 50 ml LB medium containing
tetracylcine at 37.degree. C. These cells diluted 1:100 in a fresh
LB medium containing tetracylcine and were grown at 37.degree. C.
until the OD 600 was between 0.4-0.6. IPTG was added to a final
concentration of 1 mM the cells were induced for at least 3 hours.
The cells were pelleted by centrifugation and resuspended in 100
.mu.l binding buffer containing 100 mM Hepes (pH 7.5) and 10 mM
imidazole. Cells were broken by sonication and centrifuged to
remove unbroken cells and cell debris. The supernatant was used
subsequent for purification experiments described below. In some
cases, rather than using sonication to disrupt the cells, cells
were disrupted by lysis with various detergents commonly used to
lyse bacterial cells. The use of the detergents did not interfere
with isolation of proteins.
[0150] Purification of Polypeptides Under Non-Denaturing
Conditions
[0151] Nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles (3 mg), prepared as described above, were combined with
sonicated cells containing his-tag protein and mixed by pipetting
or by shaking for approximately 1-5 minutes. The tubes were placed
on a magnetic stand to separate the particles from the supernatant,
and the supernatant was removed. The particles were washed three
times with 150 .mu.l binding buffer. His-tag proteins bound to the
particles were eluted with elution buffer (100 mM Hepes pH 7.5 and
0.1 to 0.5 M imidazole). Protein concentrations were measured using
Pierce protein assay system, and the protein was analyzed by
SDS-PAGE (FIG. 6).
[0152] FIG. 6 shows the separation using the particles of
his-RNaseHI from other proteins in the cell lysate. Lane 1, lysate
not contacted with the particles; lane 2 flow through of lysate
contacted with particles; lane 3, proteins eluted from particles
with 0.5 M imidazole; and lane 4, size marker.
[0153] Purification of Polypeptide Under Denaturing Conditions
[0154] Before sonication or cell lysis using lysis buffer, urea or
guanidine-HCl was added to cells to give a final concentration of 6
M. 30 ul of nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles was added to the cell lysate. The particles were mixed by
pipetting or by shaking for approximately 1-5 minutes. The
particles and supernatant were separated by placing the tubes on a
magnetic stand and the supernatant was removed. The particles were
washed three times (150 .mu.l each) with binding buffer containing
6 M urea or guanidine-HCl. His-tag proteins bound to the particles
were eluted with elution buffer (100 mM Hepes pH 7.5, 0.1-0.5 M
imidazole and 6 M urea or guanidine-HCl). The purified protein was
analyzed by SDS-PAGE or functional assay. Protein concentrations
were measured using Pierce protein assay system. The presence of 6
M urea or guanidine-HCl did not interfere with binding or elution
of his-tagged proteins (data not shown).
Example 11
[0155] Purification and Separation of Hemoglobin Using
Nickel/Zinc/Copper/Cobalt (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles
[0156] Nickel 3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl
magnetic silica particles, copper(II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, cobalt
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, and zinc
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were prepared as described above. Rabbit reticulocyte
lysate (Promega Corporation) (200 .mu.l) was spiked with 25 .mu.l
of purified his-luciferase proteins. A 50 .mu.l-aliquot of the
lysate was added to each of four separate tubes containing 10 mg of
nickel, copper, zinc, or cobalt
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. The particles were mixed with the lysate for 1-5 minutes
by pipetting or shaking. The tubes were placed on a magnetic stand
to separate the particles and supernatant, and the supernatant was
removed. The particles were washed three times (150 .mu.l each)
with binding buffer containing 100 mM Hepes (pH 7.5). The proteins
were eluted with 100 .mu.l elution buffer (100 mM Hepes pH 7.5 and
0.1 or 0.5 M imidazole) and analyzed by SDS-PAGE (FIG. 7). The
results indicate that hemoglobin binds to nickel, copper, zinc, or
cobalt 3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic
silica particles, and that these particles can be used to separate
hemoglobin from proteins that do not bind to the particles.
Example 12
Separation of Target from Non-Target Polypeptides Using Ni (II)
3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic silica
particles and 3-aminopropyl magnetic silica particles
[0157] (a) Purification of Target Protein by Pre-Treatment of the
Target Containing Mixture with an Aminopropyl-Modified Solid
Support
[0158] A cell lysate (100 .mu.l) of E. coli JM109 expressing
His-RNaseHI was prepared by sonicating the cells in a binding
buffer containing 20 mM Tris (pH 7.5), 0.5 M NaCl, and 20 mM
imidazole. The lysate was combined with 3-aminopropyl magnetic
silica particles (50 mg), mixed by pipetting 10 times, and
incubated for 2 minutes. The supernatant was separated from the
3-aminopropyl magnetic silica particles and mixed with 3 mg of Ni
(II) 3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic
silica particles by pipetting (10.times.) for 2 minutes. The
supernatant, which contained primarily non-target proteins, was
removed and discarded. The Ni (II)
3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl particles were
washed 3 times with 150 .mu.l of a buffer containing 20 mM Tris (pH
7.5), 0.5 M NaCl, and 20 mM imidazole. The H is RNaseHI was then
eluted with an elution buffer (100 .mu.l) containing 20 mM Tris (pH
7.5), 0.5 M NaCl, and 0.5 M imidazole. The samples were analyzed by
gel electrophoresis (FIG. 8). With reference to FIG. 8, lane 4
contains a marker, lane 5 contain the unfractionated bacterial
lysate, lane 6 contains the flow-through solution from
3-aminopropyl magnetic particles, lane 7 contains the flow-through
from Ni (II) 3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl
magnetic silica particles, lane 8 contains the 0.5 M imidazole
eluate from the Ni (II)
3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic silica
particles, lane 9 contains the flow through fraction from
3-aminopropyl magnetic silica particles, lane 10 contains the flow
through from Ni (II)
3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic silica
particles, and lane 11 contains the 0.5 M imidazole eluate from the
Ni (II) 3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic
silica particles.
[0159] (b) Purification of Target Protein by Posttreatment of the
Target-Containing Mixture with an Aminopropyl-Modified Solid
Support
[0160] A cell lysate of E. coli JM109 expressing his-tagged
luciferase was prepared by sonicating JM109 cells in a binding
solution containing 20 mM Tris (pH 7.5), 0.5 M NaCl, and 20 mM
imidazole. The lysate (100 ul) was mixed with 3 mg of Ni (II)
3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic silica
particles by pipetting (10.times.) for 2 minutes. The particles
were separating from the binding solution using a magnet and washed
with three times with 150 .mu.l of the binding solution. The target
protein was eluted by adding 100 .mu.l of 20 mM Tris (pH 7.5), 0.5M
NaCl, and 0.5 M imidazole, pH 7.5. The eluted target protein was
further purified from residual background polypeptides by mixing
with 3 mg of 3-aminopropyl magnetic silica particles for 2 minutes
and separating the target-containing supernatant from the
particles. The samples were analyzed by gel electrophoresis
(results not shown).
[0161] The results indicate that 3-aminopropyl magnetic silica
particles, used in conjunction with Ni (II)
3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic silica
particles, facilitate removal of contaminating proteins.
Example 13
Method for the Quantitation of Polypeptides Using Nickel (II)
3[[[Bis(carboxymethyl)amino]acetyl]amino-propyl magnetic silica
particles
[0162] Aliquots of lysate from bacteria expressing his-luciferase
(100 .mu.l) were placed into three Eppendorff tubes and mixed with
50 .mu.l nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, prepared as described above, by pipetting for 1-2
minutes. Particles to which no lysate was added were used as a
control. The particles were washed with 1 ml of 100 mM Hepes (pH
7.5). An aliquot (100.mu.) of 1% Coomassie blue in 100 mM Hepes (pH
7.5) was added to the washed particles and to the control
particles. The particles were washed extensively with 100 mM Hepes
pH 7.5 until the wash buffer was clear. The his-tagged luciferase
was eluted with 0.1 M imidazole, 0.2 M imidazole, or 0.5 M
imidazole and the collected eluate was photographed (FIG. 9A). The
absorbance was measured by a spectrophotometer at a wavelength of
595 nm (FIG. 9B). The amount of labeled protein recovered was
positively correlated with the concentration of imidazole used to
elute the protein. In a parallel experiment, nickel agarose beads
(Qiagen) were substituted for the .mu.l nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, the labeled proteins eluted with 0.5 M imidazole, and
the eluate photographed (FIG. 9C).
[0163] In a similar experiment, JM109 cells expressing
His-methionyl tRNA synthetase were grown to an OD.sub.600 and
induced with 1 mM IPTG. Aliquots of the cultures were collected at
half hour intervals through three hours post-induction, and used to
prepare lysates that were treated as described above in the
preceding paragraph. FIG. 23A is a photograph of eluted, Coomassie
stained proteins, illustrating that recovery of labeled protein is
positively correlated with the time post-induction. FIG. 23 B is a
graph plotting cell growth (as measured by OD.sub.600) and protein
concentration (as measured by A.sub.595 of Coomassie stained
proteins) as a function of time post-induction. FIG. 23 C is an
SDS-PAGE gel of the purified protein in the samples.
[0164] Purified his-tagged proteins were contacted with nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles and treated with BODIPY dye. The proteins/particles were
separated by SDS-PAGE and visualized using fluorescent imager
scanning (FIG. 24). With reference to FIG. 24, lane 1 contains
His-firefly luciferase (62 kDa); lane 2 contains His-Renilla
luciferase (36 kDa); lane 3 contains His-RNasin inhibitor (45 kDa);
and lane 4 contains His-methionyl tRNA synthetase (76 kDa).
[0165] The experiment describes a method for quantitating proteins
using in-particle labeling of proteins with dyes. Imidazole
interferes with protein assays such as Bradford or BCA and must be
removed by dialysis prior to measuring protein concentrations by
those methods. In contrast, because imidazole not interfere with
this assay, protein concentrations in samples can be evaluated
directly without first dialyzing the samples.
Example 14
[0166] Method of Detecting Fluorescently Labeled Polypeptides
[0167] Copper (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles or nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were prepared as described above. The particles were
washed once with 100 mM Hepes (pH 7.5). Aliquots (100 .mu.l) of a
bacterial lysate expressing his-luciferase or a BSA (10 mg/ml) in
100 mM Hepes (pH 7.5) were placed into separate Eppendorff tubes. A
50-.mu.l aliquot of 10% (w/v) nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles was added to the lysate in each tube and 50 .mu.l 10%
(w/v) copper (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles was added to the BSA in each tube and mixed by pipetting
for 1-2 min. The particles were washed with 1 ml of 100 mM Hepes pH
7.5. Then, 100 .mu.l of Fluorscein or Bodipy was dissolved in 60%
Acetonitrile in 100 mM Hepes (pH 7.5) and added to the washed
particles as well as to the control particles. The particles were
washed 3.times. with 100 mM Hepes (pH 7.5) or until the free,
unbound Fluorescein or Bodipy was removed. The bound polypeptides
were eluted with 0.5 M imidazole. Polypeptides were detected by
running the samples on SDS-PAGE followed by UV detection on a
fluoroimager. As can be seen in FIG. 10A, his-luciferase labeled
with Bodipy (lane 1) or Fluorescein (lane 2) was detectable. As can
be seen in FIG. 10B, BSA labeled with Bodipy (lane 1) or
Fluorescein (lane 2) was detectable.
[0168] The results indicate that proteins can be labeled with
fluorescent dyes while the proteins are attached to the particles.
This facilitates removal of free dye from the sample and affords
rapid detection and quantitation of polypeptides.
Example 15
[0169] Isolation of tRNA Synthetase Using Nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles
[0170] Plasmid DNA encoding his-methionyl tRNA synthetase was
expressed in an in vitro translation reaction in S-30 (Promega
Corporation). The reaction mixtures contained 8 .mu.g plasmid DNA,
5 .mu.g Bodipy f-Met tRNA, amino acids (25 .mu.l), S-30 premix (100
.mu.l), and S-30 extract (75 .mu.l). Reactions were performed at
37.degree. C. for one hour. Each reaction mixture was combined with
3 mg of nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles and mixed by pipetting. The particles were washed three
times with 100 mM Hepes (pH 7.5). Material bound to the particles
was eluted with 10 mM ammonium acetate. As a control, particles
were contacted with the reaction mixture and washed, but not
treated with ammonium acetate. The ammonium acetate eluate and the
control particles were treated with 100 .mu.l of a protein
denaturation buffer and placed at 70.degree. C. for 5 minutes, and
analyzed by SDS-PAGE (FIG. 11). With reference to FIG. 11, lane 1
contains untreated lysate; lane 2 contains the flowthrough of
lysate applied to the particles; lane 3 contains the ammonium
acetate eluate; and lane 4 contains particles not treated with
ammonium acetate. The results illustrate that tRNA binds tightly to
the particles and that a portion of the tRNA can be eluted from the
particles using an elution buffer containing 10 mM ammonium
acetate.
Example 16
Purification of Cell-Free Expressed His-Tagged GFP
[0171] Prokaryotic in vitro transcription/translation reactions to
express his-tagged GFP were conducted in 0.5 ml T7-S30 reaction
volumes with 15 .mu.g DNA template (pGFP-HIS) using the Rapid
Translation System R.sub.T 500 (Roche) according to the
manufacturer's instructions and incubated at 30.degree. C. for 20
hours using the RTS 500 instrument (Roche). When used,
FluoroTect.TM. or Bodipy.RTM.-fMet-tRNA was included at a
concentration of 1 .mu.g/50 .mu.l of T7-S30 reaction.
[0172] The his-tagged GFP was purified by mixing the reaction
mixtures with nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. The particles were washed twice with 100 mM Hepes (pH
7.5) and 10 mM imidazole. The particles were then washed with 30%
methanol and eluted with 50% acetonitrile and 0.1% trifluoroacetic
acid in water.
[0173] The samples (5 .mu.l, or in the case of wash or elution
samples, 10 .mu.l) were mixed with 20 .mu.l of 4.times.SDS sample
buffer and were run on 4-20% Novex Tris/Glycine gels, stained with
Gel-Code and the fluorescent images captured with a digital camera
(FIG. 12). With reference to FIG. 12, lanes 1 and 10 contain a size
marker, lane 2 contains the S-30 lysate without DNA, lane 3
contains the S-30 lysate with plasmid DNA, lane 4 contains the S-30
lysate with plasmid DNA and Bodipy-fMet tRNA, lane 5 contains the
S-30 lysate with plasmid DNA, lane 4 contains the S-30 lysate with
plasmid DNA and fluorotect tRNA, lane 6 contains the S-30 lysate
without plasmid DNA eluted with 0.1% trifluoroacetic acid in water,
and lanes 7-9 contain S-30 lysate with plasmid DNA eluted with 50%
acetonitrile and 0.1% trifluoroacetic acid in water.
[0174] The results indicate that nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles can be used to purify his-tagged proteins expressed in
cell free expression systems.
Example 17
[0175] In-Particle Functional Assay of tRNA Synthetase Activity
[0176] The E. coli strain JM109 expressing his-methionyl tRNA
synthetase was grown overnight at 37.degree. C. in 50 ml LB medium
containing tetracycline. A 15-ml aliquot of the overnight culture
was added to 3 L LB medium and was grown at 37.degree. C. When the
culture reached the OD.sub.600 between 0.4-0.6, IPTG was added to a
final concentration of 1 mM and the cells were induced for at least
3 hours. Cells were pelleted by centrifugation and resuspended in
10 ml of 10 mM Hepes buffer (pH 8.0) and 5 mM MgCl.sub.2 (buffer
A). The sample was sonicated and pelleted by centrifugation. The
supernatant containing his-methionyl tRNA synthetase was mixed with
10 ml of nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles and incubated at 4.degree. C. for 1 hr. The particles
were washed five times with buffer A and used in the functional
assay of bound tRNA synthetase, as described below. Unbound
purified tRNA synthetase used in the assay was obtained by eluting
bound protein from a sample of the particles with 0.5 M imidazole
and dializing the eluate to remove the imidazole.
[0177] The activity of bound tRNA was assayed by incubating 84
.mu.l of the particles containing bound methionyl tRNA synthetase,
prepared as described in the preceding paragraph, 14.4 .mu.l folic
acid (0.01 M), 8.0 .mu.l .sup.35S Met, 7.2 .mu.l (2 M) NaCl, 12.0
.mu.l (1 mM) Met, 144.0 .mu.l (1 M) Hepes (pH 8.0), 14.4 .mu.l (0.1
M) MgCl.sub.2, 57.6 .mu.l (0.1 M) ATP, 14.4 .mu.l (0.01 M) CTP,
14.4 .mu.l (0.1 M) DTT and 469.6 .mu.l sterile double distilled
water at 37.degree. C. for 15 minutes. Included as a control was
free his-methionyl tRNA synthetase prepared as described above. A
1201 aliquot of 10% TCA was added and the mixtures incubated on ice
for 15 minutes. The samples were filtered through 0.2 .mu.m glass
microtitre filters (Whatman), washed with 10% TCA, and washed with
10% ethanol. The filters were dried and counted in a scintillation
counter. The results are presented in 13, which shows that the
activity of the bound his-methionyl tRNA synthetase approaches that
of the free, purified his-methionyl tRNA synthetase.
Example 18
Preparation of Proteins for Mass Spectrometer Analysis Using
Modified Pipette Tips
[0178] Pipette tips for mass spectrometer analysis are prepared
using Promega 200 Barrier Tip 200-.mu.l plastic pipette tips
(Promega Corp., Madison, Wis.). The tips are loaded with 50-100
.mu.L of nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles or copper (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, prepared by modifying silica, as described above, using
silica having a diameter in the range of 100 .mu.m to 150 .mu.m
(Sigma-Aldrich Corp., Milwaukee, Wis.). The particles are
introduced into pipette tips (FIG. 14). Prior to use, the particles
in the tips are washed three times with 1 ml of 100 mM Hepes buffer
(pH 7.5) with 10 mM imidazole. The pipette tips may include plastic
or glass pipette tips having a capacity in the range of 10 .mu.l to
1 ml. The amount of resin in the tip may be adjusted according the
sample volume or the amount of protein to be purified.
[0179] Protein Purification/Fractionation
[0180] For complex protein analysis, 5 .mu.l of rabbit reticulocyte
lysate (Promega) is mixed with 195 .mu.l of 100 mM Hepes (pH 7.5).
A 50 .mu.l portion of the sample is transferred to a pipette tip,
prepared as described above, containing 100 .mu.l copper (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. The sample is mixed by pipetting the sample into and out
of the pipette tip 5 or 6 times. Proteins are allowed to bind to
the particles by for 1-2 minutes. The particles are washed three
times with 1 ml of 100 mM Hepes (pH 7.5). Bound proteins are eluted
with 100 .mu.l 0.1% TFA in water. The eluted samples are dried in
Speed Vac and analyzed in a mass spectrometer (HT
Laboratories).
[0181] To isolate his-tagged proteins, 50 .mu.l of a bacterial
lysate containing his-tagged proteins is transferred into a pipette
tip containing 100 .mu.l nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. The sample is mixed with the particles by pipetting in
and out at least 5 or 6 times. The particles are washed three times
with 1 ml binding buffer. The protein is eluted with 100 .mu.l 0.1%
TFA in water. The eluted samples are dried in Speed Vac and
analyzed in a mass spectrometer (HT Laboratories).
Example 19
Sequential Multidimensional Polypeptide Fractionation and
Separation Using Immobilized Metal Chelated Chromatography
(IMAC)
[0182] Copper 3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl
magnetic silica particles, cobalt
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, and zinc
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were prepared as described above. Gallium
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles and iron
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were prepared by removing the liquid from one-ml aliquots
of 10% 3-[[[Bis(carboxymethyl)amino]acetyl]amino]-propyl magnetic
silica particles by placing onto a magnetic stand, and adding 1 ml
of 250 mM gallium (III) nitrate in water or 1 ml 250 mM iron (III)
sulfate in water to the particles. The particles and metal
solutions were mixed well by pipetting. The particles were
separated from the metal solution by placing onto a magnetic stand
and the metal solution removed. A second 1 ml aliquot of metal
solution was mixed with the particles in each tube, incubated for 2
minutes, and the metal solution removed by placing onto a magnetic
stand. The particles were washed four times with 1 ml MilliQ water,
and then washed once with 100 mM Hepes (pH 7.5). Protein
fractionation was performed as described below.
[0183] Binding and Elution of Complex Mixture of Proteins.
[0184] Aliquots of rabbit reticulocyte lysate (5 .mu.l) (Promega
Corp.) were diluted with 195 .mu.l of 100 mM Hepes (pH 7.5).
Diluted lysate was mixed with 100 .mu.l of
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles or with nickel, cobalt, copper, zinc, iron, or gallium
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles in Eppendorf tubes by pipetting for 1-2 minutes. The
tubes were then placed onto a magnet, the supernatant was removed,
and the particles washed three times with 1 ml of 100 mM Hepes (pH
7.5). Bound proteins were eluted from the particles with 0.5 M
imidazole and were analyzed by SDS-PAGE (FIG. 15). Lane 1, size
marker; lane 2, unfractionated rabbit reticulocyte lysate; lanes
3-9 flow through fractions from
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, nickel, cobalt, copper zinc, iron (III), or gallium
(III) 3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic
silica particles, respectively; lanes 10-16, imidazole eluate from
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, nickel, cobalt, copper zinc, iron (III), or gallium
(III) 3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic
silica particles, respectively. The results show that cobalt and
copper 3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic
silica particles bind relatively tightly to most proteins in the
rabbit reticulocyte lysate under the conditions employed.
[0185] From 1-20 .mu.l of rabbit reticulocyte lysate was combined
with copper 3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl
magnetic silica particles and processed as described above (FIGS.
16A and 16B). Lane 1, size marker; lane 2, unfractionated rabbit
reticulocyte lysate; lanes 3-6, flow through fraction from 3, 5,
10, or 20 .mu.l of lysate, respectively, fractionated on copper
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles; lanes 7-10, imidazole eluate from 3, 5, 10, or 20 .mu.l
of lysate on copper
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles. The protein concentration for each fraction was
determined by the Bradford method. FIG. 16B shows the absorbance
(595 nm) of the flow through or eluate as a function of lysate
volume fractionated.
[0186] CHO cell lysate was prepared by suspending 8.times.10.sup.6
cells in 1 ml of 100 mM Hepes (pH 7.5) and breaking the cells by
freeze thawing. The cells were centrifuged, and the supernatant
reserved. A supernatant aliquot of 100 .mu.l was used for each
experiment. Binding, washing, elution and analysis were done as
explained for rabbit reticulocyte lysate. Results are shown in FIG.
17. With reference to FIG. 17A, lane 1 contains unfractionated CHO
cell lysate; lane 4 contains protein molecular weight marker; lanes
2, 3, 5, and 6 contain flow through from 3, 5, 10 or 20,
respectively; lanes 7-9 contain the imidazole eluate from 3, 5, or
10 .mu.l CHO lysate.
[0187] Wheat germ lysate (Promega) was also used for binding
studies. 50 .mu.l of wheat germ lysate was added to 50 .mu.l of 100
mM Hepes (pH 7.5) buffer and was used for the experiment. Binding,
washing, elution and analysis were done as explained for rabbit
reticulocyte lysate. Results are shown in FIG. 18.
[0188] Sequential Multidimensional Separation of Proteins
[0189] Aliquots of rabbit reticulocyte lysate (5 .mu.l) (Promega
Corp.) were diluted with 195 .mu.l of 100 mM Hepes (pH 7.5).
Diluted lysate was mixed with 100 .mu.l copper
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles in Eppendorf tubes by pipetting for 1-2 minutes. The
tubes were then placed onto a magnet, the supernatant was removed,
and the particles washed three times with 1 ml of 100 mM Hepes (pH
7.5). Proteins were eluted by sequentially treating the particles
with 1, 5, 10, 20 and 50% acteonitrile. The particles were then
treated with double distilled water, followed by eluting with 0.1
and 1% trifluoroacetic acid (TFA). All these samples were analyzed
by SDS-PAGE. Results are shown in FIG. 19.
[0190] In a separate experiment, proteins were first eluted with
100, 200, 500 or 1000 mM imidazole, followed by the elution of the
same particles with buffers of pH 8.5, 9.5, 10.5, or 12.5. Samples
were analyzed by SDS-PAGE and results are shown in FIG. 20.
Example 20
Separation of Phosphoproteins
[0191] Iron (III) and gallium (III)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles, prepared as described above, were equilibrated with 100
mM Hepes (pH 7.5).
[0192] A solution of ovalbumin (Sigma) containing 10 mg/ml in 100
mM Hepes (pH 7.5) was prepared. Aliquots (100 .mu.l) of the
solution were added to Iron (III) and gallium (III)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles and mixed well by pipetting. Nickel (II)
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles and uncharged
3-[[[Bis(carboxymethyl)amino]-acetyl]amino]-propyl magnetic silica
particles were included as controls. After binding, particles were
washed three times with 100 mM Hepes (pH 7.5) buffer. Bound protein
was eluted using 2% ammonium hydroxide. The samples were analyzed
by SDS-PAGE (FIG. 21). In parallel experiments, rabbit reticulocyte
(diluted 1:1 with 100 mM Hepes (pH 7.5)) was used, and the
fractions analyzed by SDS-PAGE (FIG. 22).
Example 21
Screening Expression Libraries for Membrane Proteins
[0193] The method of isolation of membrane proteins described in
Examples 7 and 8, above, will be used to screen libraries for
expression of membrane proteins. Pools of c-DNA clones (50-100
clones per pool) will be used as templates for small-scale
transcription/translation reactions to generate proteins in
presence of canine or HeLa microsomal membrane in a 96 well format.
Amine-modified silica magnetic particles will be added to the
reaction mix to capture the membrane vesicles and any associated
membrane proteins.
[0194] The foregoing description of the invention is exemplary for
purposes of illustration and explanation. It will be apparent to
those skilled in the art that changes and modifications are
possible without departing from the spirit and scope of the
invention. It is intended that the following claims be interpreted
to embrace all such changes and modifications.
* * * * *