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BACKGROUND OF THE INVENTION
This invention relates to a method and system for separating a selected
molecule from a heterogeneous mixture of molecules. More particularly, the
invention relates to separating a selected molecule from a heterogeneous
mixture of molecules by reversibly coupling the selected molecule to a
motor protein such that the motor protein can transport the selected
molecule away from the heterogeneous mixture by moving on microtubules
immobilized in a separation device.
One of the fundamental processes occurring in biological cells is active
transport on a sub-micrometer scale. The simplest eukaryotic cell contains
thousands of components that must be processed, packaged, sorted, and
delivered to specific locations at specific times within the cell. These
essential transport processes are carried out by motor proteins that
travel along microtubules reaching into every corner of the cell. Motor
proteins can be conceptualized as biological machines that transduce
chemical energy into mechanical forces and motion.
The motor protein, kinesin, was discovered in 1985 in squid axoplasm. R. D.
Vale et al., Identification of a Novel Force-generating Protein, Kinesin,
Involved in Microtubule-based Motility, 42 Cell 39-50 (1985). In the last
few years, it has been discovered that kinesin is just one member of a
very large family of motor proteins. E.g., S. A. Endow, The Emerging
Kinesin Family of Microtubule Motor Proteins, 16 Trends Biochem. Sci. 221
(1991); L. S. B. Goldstein, The Kinesin Superfamily: Tails of Functional
Redundancy, 1 Trends Cell Biol. 93 (1991); R. J. Stewart et al.,
Identification and Partial Characterization of Six Members of the Kinesin
Superfamily in Drosophila. 88 Proc. Nat'l Acad. Sci. USA 8470 (1991).
Other motor proteins include dynein, e.g. M.-G. Li et al., Drosophila
Cytoplasmic Dynein, a Microtubule Motor that is Asymmetrically Localized
in the Oocyte, 126 J. Cell Biol. 1475-1493 (1994), and myosin, e.g. T. Q.
P. Uyeda et al., 214 J. Molec. Biol. 699-710 (1990). Kinesin, dynein, and
related proteins move along microtubules, whereas myosin moves along actin
filaments. It has now become apparent that eukaryotic cells use motor
proteins to mediate numerous transport requirements. In addition to its
motor activity, kinesin is also a microtubule-activated adenosine
triphosphatase (ATPase).
Kinesin is composed of two heavy chains (each about 120 kDa) and two light
chains (each about 60 kDa). The kinesin heavy chains comprise three
structural domains: (a) an amino-terminal head domain, which contains the
sites for ATP and microtubule binding and for motor activity; (b) a middle
or stalk domain, which may form an .alpha.-helical coiled coil that
entwines two heavy chains to form a dimer; and (c) a carboxyl-terminal
domain, which probably forms a globular tail that interacts with the light
chains and possibly with vesicles and organelles. Kinesin and kinesin-like
proteins are all related by sequence similarity within an approximately
340-amino acid region of the head domain, but outside of this conserved
region they show no sequence similarity.
The motility activity of purified kinesin on microtubules has been
demonstrated in vitro. R. D. Vale et al., Identification of a Novel
Force-generating Protein, Kinesin, Involved in Microtubule-based Motility,
42 Cell 39-50 (1985). Further, fulllength kinesin heavy chain and several
types of truncated kinesin heavy chain molecules produced in E. coli are
also capable of generating in vitro microtubule motility. J. T. Yang et
al., Evidence that the Head of Kinesin is Sufficient for Force Generation
and Motility In Vitro, 249 Science 42-47 (1990); R. J. Stewart et al.,
Direction of Microtubule Movement is an Intrisic Property of the Motor
Domains of Kinesin Heavy Chain and Drosophila NCD Protein, 90 Proc. Nat'l
Acad. Sci. USA 5209-5213 (1993). The kinesin motor domain has also been
shown to retain motor activity in vitro after genetic fusion to several
other proteins including spectrin, J. T. Yang et al., The Head of Kinesin
is Sufficient for Force Generation and Motility In Vitro, 249 Science 42
(1990), glutathione S-transferase, R. J. Stewart et al., Direction of
Microtubule Movement is an Intrinsic Property of the NCD and Kinesin Heavy
Chain Motor Domains, 90 Proc. Nat'l Acad. Sci. USA 5209 (1993), and biotin
carboxyl carrier protein, E. Berliner, Microtubule Movement by a
Biotinated Kinesin Bound to a Streptavidin-coated Surface, 269 J. Biol.
Chem. 8610 (1994).
Similarly, methods have been developed for manipulation of microtubules.
Microtubules can be routinely reassembled in vitro from tubulin purified
from bovine brains. The nucleation, assembly, and disassembly reactions of
microtubules have been well characterized. L.U. Cassimeris et al., Dynamic
Instability of Microtubules, 7 Bioessays 149 (1988). More recently,
considerable progress has been made toward producing recombinant tubulin
in yeast. A. Davis et al., Purification and Biochemical Characterization
of Tubulin from the Budding Yeast Saccharomyces cerevisiae, 32
Biochemistry 8823 (1993).
Separation of selected molecules from complex mixtures of molecules is of
great importance in chemical, pharmaceutical, biotechnological,
health-related and medical, and many other industries. Great amounts of
time and money are spent on performing such separations. There is also an
interest in instrument miniaturization driven by potential for
substantially decreased analysis time, decreased reagent volumes and cost,
decreased analyte volumes, integration of analytical techniques in a
single device, and the economy of batch fabrication of complex devices.
In view of the foregoing, it will be appreciated that providing a method of
separating a selected molecule from a heterogeneous mixture of molecules
by reversibly coupling the selected molecule to a motor protein for
transport on microtubules immobilized in a separation device would be a
significant advancement in the art.
BRIEF SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for separating
a selected molecule from a heterogeneous mixture of molecules.
It is also an object of the invention to provide a method for separating a
selected molecule from a heterogeneous mixture of molecules by reversibly
coupling the selected molecule to a motor protein, which transports the
selected molecule on immobilized microtubules.
It is another object of the invention to provide a microfabricated device
comprising immobilized microtubules for performing separations using a
motor protein reversibly coupled to a selected molecule to be isolated.
It is still another object of the invention to provide a separation system
that recognizes, separates, and detects selected molecules on a single
micromachined chip.
These and other objects are accomplished by providing a method for
separating a selected molecule from a heterogeneous mixture of molecules
comprising:
(a) providing a separation device comprising a loading reservoir and a
receiving reservoir coupled by a channel having immobilized to a surface
thereof a plurality of microtubules aligned substantially parallel to a
longitudinal axis of the channel;
(b) placing an aqueous solution comprising the heterogeneous mixture of
molecules in the loading reservoir;
(c) adding a motor-ligand composition and an effective amount of ATP to the
aqueous solution, wherein the motor-ligand composition comprises
(i) a motor protein capable of attaching to the immobilized microtubules
and moving therealong in the presence of ATP as a source of chemical
energy, and
(ii) a ligand coupled to the motor protein, wherein the ligand is capable
of selectively binding the selected molecule,
such that the ligand selectively binds the selected molecule and the motor
protein attaches to the immobilized microtubules and transports the bound
selected molecule therealong to the receiving reservoir; and
(d) removing the selected molecule from the receiving reservoir.
Preferably, the motor protein comprises the N-terminal 410 amino acid
residues of kinesin. In one illustrative embodiment, the ligand comprises
an oligonucleotide having a nucleotide sequence capable of hybridizing to
a target site on the selected molecule. A preferred oligonucleotide has a
nucleotide sequence capable of hybridizing to a phage .lambda. cos site,
wherein the target site comprises a phage .lambda. cos site. In another
preferred embodiment, the ligand comprises an oligonucleotide and the
method further comprises providing an adaptor oligonucleotide comprising a
first hybridization site and a second hybridization site, wherein the
ligand is capable of hybridizing to the first hybridization site and the
second hybridization site is capable of hybridizing to a target site on
the selected molecule.
In another preferred embodiment, the ligand comprises a peptide, such as
streptavidin, protein A, or an immunoglobulin such as a single chain
antibody. With a streptavidin, the ligand will bind any biotinylated
molecule, or a biotinylated bead can be used to simultaneously bind a
plurality of motor-ligand compositions and a plurality of selected
molecules conjugated to streptavidin.
In another preferred embodiment, the method further comprises, prior to
removing the selected molecule from the receiving reservoir, detecting the
presence of the selected molecule in the receiving reservoir. For example,
detection of a nucleic acid, protein, or other selected molecule can be
with an appropriate fluorescent dye.
The invention also comprises aligning the microtubules in the channel of
the separation device. Preferred methods of aligning the microtubules
include flow alignment, nucleating with centrosomes or axoneme fragments,
and fletching.
A preferred separation device is a micrometer-scale device wherein the
loading reservoir, receiving reservoir, and channel are micromachined into
a substrate.
Another aspect of the invention is a system for separating a selected
molecule from a heterogeneous mixture of molecules in aqueous solution
comprising:
(a) a separation device comprising a loading reservoir and a receiving
reservoir coupled by a channel having immobilized to a surface thereof a
plurality of microtubules aligned substantially parallel to a longitudinal
axis of the channel;
(b) a motor-ligand composition comprising
(i) a motor protein capable of attaching to the immobilized microtubules
and moving therealong in the presence of ATP as a source of chemical
energy, and
(ii) a ligand coupled to the motor protein, wherein the ligand is capable
of selectively binding the selected molecule;
(c) an effective amount of ATP for providing chemical energy to the motor
protein for supporting movement thereof along the immobilized microtubules
.
BRIEF DESCRIPTION OF THE SEVERAL VIEW OF THE DRAWINGS
FIG. 1 shows a diagrammatic representation of a nerve cell or neuron.
FIG. 2 shows a diagrammatic representation of a neuronal vesicle with
kinesin molecules bound to the surface thereof.
FIG. 3 shows a top schematic view of a microfabricated device according to
the present invention.
FIG. 4 shows an elevation side view of a microfabricated device according
to the present invention.
FIG. 5 shows a cross-section of the microfabricated device of FIG. 4.
FIGS. 6A-E show immobilization of microtubules on the surface of a
microchannel in a microfabricated device according to the present
invention.
FIGS. 7A-C show, respectively, methods of aligning microtubules in the
microchannel of a microfabricated device by flow alignment, nucleating
with centrosomes or axoneme fragments, and fletching.
FIG. 8 shows a schematic diagram of a detection system coupled to a
separation system according to the present invention.
FIG. 9 shows a construction map of a plasmid, p-MON-kin-sav, for expression
of a kinesin-streptavidin fusion protein.
DETAILED DESCRIPTION
Before the present method and system for separating a selected molecule
from a heterogeneous mixture of molecules are disclosed and described, it
is to be understood that this invention is not limited to the particular
configurations, process steps, and materials disclosed herein as such
configurations, process steps, and materials may vary somewhat. It is also
to be understood that the terminology employed herein is used for the
purpose of describing particular embodiments only and is not intended to
be limiting since the scope of the present invention will be limited only
by the appended claims and equivalents thereof.
It must be noted that, as used in this specification and the appended
claims, the singular forms "a," "an," and "the" include plural referents
unless the context clearly dictates otherwise. Thus, for example,
reference to a separation system containing "a microtubule" includes a
system containing two or more of such microtubules, reference to "a
motor-ligand composition" includes reference to two or more of such
motor-ligand compositions, and reference to a separation system containing
"a channel" includes reference to two or more of such channels.
In describing and claiming the present invention, the following terminology
will be used in accordance with the definitions set out below.
As used herein, "micromachining," "micromachined," and similar terms refer
to the processes used to create micrometer-sized structures with primarily
mechanical functions on a glass, silicon, silica, or photoreactive
polymer-coated chip or other suitable substrate. The processes of
micromachining are based on techniques developed in the microelectronics
industry to create layered structures in integrated circuits, e.g.
photolithography and film deposition procedures. In a preferred embodiment
of the present invention, the dimensions of a microchannel connecting a
loading reservoir and a receiving reservoir are about 125 .mu.m in length
by about 25 .mu.m in width by about 10 .mu.m in depth, but the dimensions
of such microchannels are limited only by functionality. The dimensions of
the loading and receiving reservoirs are not considered to be critical and
are also limited only by functionality. The microchannel is constructed of
sufficient length such that the motor-ligand composition can transport a
selected molecule from the loading reservoir to the receiving reservoir
before contaminating molecules reach the receiving reservoir by diffusion.
Kinesins move at a rate of about 60 .mu.m/min. Diffusion of undesirable
molecules can be retarded by application of an electrical field and/or
increasing the viscosity of the liquid medium, and the like.
As used herein, "hybridization," "hybridizing," and similar terms refers to
forming double-stranded nucleic acid molecules by hydrogen bonding of
complementary base pairs, as is well known in the art. A person skilled in
the art will recognize that a certain amount of mismatching is permitted
under certain circumstances such that hybridization will still occur.
Further, the conditions of hybridization can be manipulated by varying the
lengths and GC ratios of complementary sequences that are to be
hybridized, the amount of mismatching, the monovalent salt concentration,
the presence of certain solvents such as formamide, and the temperature,
according to principles well known in the art, such as are described in J.
Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989); T.
Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); F. Ausubel
et al., Current Protocols in Molecular Biology (1987), hereby incorporated
by reference.
As used herein, "motor protein" means a protein that transduces chemical
energy into mechanical forces and motion. Preferred motor proteins for the
present invention are kinesin and related proteins, such as ncd, S. A.
Endow et al., Mediation of Meiotic and Early Mitotic Chromosome
Segregation in Drosophila by a Protein Related to Kinesin, 345 Nature
81-83 (1990), hereby 11 incorporated by reference, that are highly
processive, i.e. do not readily detach from the microtubule tracks to
which they are coupled. Once such highly processive motor proteins attach
to a microtubule, there is a relatively high likelihood that they will
move for many micrometers along the microtubule before becoming detached.
Motor proteins such as myosin and dynein are considered unsuitable for use
in the present invention because they lack high processivity. Preferred
motor proteins are "double-headed," that is they are heavy chain dimers,
which in part explains their processivity. Kinesin moves toward the
plus-end of microtubules, whereas ncd moves toward the minus-end thereof.
As used herein, "ligand" refers to a moiety that reversibly binds a
selected molecule. In coordination compound chemistry, a ligand is a
molecule or anion that donates a pair of electrons to a central metal atom
to form a coordinate covalent bond between the ligand and the metal atom;
thus, the ligand binds the metal atom. "Ligand" is used more broadly
herein to refer to any moiety that reversibly binds a selected molecule
that is to be separated from a mixture of molecules. For example, a ligand
can be a single-stranded nucleic acid molecule that is adapted for and is
capable of hybridizing to a selected complementary nucleic acid molecule.
In another illustrative example, a ligand can be an antibody, Fab,
F(ab').sub.2, F(ab'), single chain antibody, or the like that is capable
of binding a selected antigen. In another illustrative embodiment, a
ligand can be a protein A molecule, which is capable of binding IgG
molecules. In another illustrative example, a ligand can be avidin or
streptavidin, which is capable of binding biotin or a biotinylated
molecule of interest.
As used herein, "motor-ligand composition" refers to a motor protein
coupled to a ligand molecule. The motor protein portion of the
motor-ligand composition is preferably derived from kinesin, ncd, or
another highly processive kinesin-related motor protein. The motor protein
portion should be double-headed, therefore it will contain at least about
the N-terminal 410 amino acid residues of the heavy chain protein. Amino
acid residues in addition to the N-terminal 410 amino acid residues can
also be present, and in this respect the length of the motor protein
molecules is limited only by functionality, but preferably the motor
protein chain contains no more than about 900 amino acid residues. Several
illustrative constructions are exemplified herein. Recombinant motor
proteins are also considered within the scope of the present invention. A
few illustrative motor-ligand compositions are described herein, but it
should be recognized that a person of skill in the art could easily
construct additional motor-ligand compositions by recombinant DNA
technology.
The ligand portion of the motor-ligand composition can be any ligand that
will selectively bind to a selected molecule to be separated from a
mixture of molecules, provided that the ligand can be coupled to the motor
protein without destroying the ability of the ligand to bind the selected
molecule or the ability of the motor protein to move on the microtubules.
For example, nucleic acids and certain proteins are preferred ligands.
Selected oligonucleotides can be coupled to a motor protein, as will be
discussed in more detail momentarily, such that the oligonucleotide is
capable of hybridizing to a selected molecule, i.e. a nucleic acid
molecule, that is to be separated in the separation process. The variety
of molecules that can be subject to such separations is extremely wide, as
will be appreciated. By way of further example, proteins such as
streptavidin, protein A, and single chain antibodies can be coupled to a
motor protein for binding a wide variety of molecules. Streptavidin is
known to bind biotin, thus any molecule that can be biotinylated, such as
DNA and proteins, can be separated with such a ligand. Protein A is known
to bind to IgG molecules. Single chain antibodies can be produced that
will bind to virtually any immunogen.
Coupling of an oligonucleotide ligand to a motor protein can be by any
method known in the art such that the motility of the motor protein
portion and ability of the oligonucleotide to hybridize are preserved. An
illustrative method of coupling an oligonucleotide to a motor protein will
be exemplified below. Coupling of a motor protein to a protein or
polypeptide ligand can also be carried out by known methods, such as
chemical coupling or, preferably, expression of a fusion protein by
recombinant DNA technology. Such recombinant DNA methods are described in
the Sambrook et al., Maniatis et al., and Ausubel references. Briefly, a
gene encoding a motor protein is spliced to a gene encoding a selected
ligand polypeptide to form a gene fusion, and then the gene fusion is
expressed in a suitable expression system such as E. coli or yeast to
produce the motor-ligand composition, which is then purified and used in
the separation system.
As used herein, "peptide" means peptides of any length and includes
proteins. The terms "polypeptide" and "oligopeptide" are used herein
without any particular intended size limitation, unless a particular size
is otherwise stated.
As used herein, "effective amount" means an amount of a source of chemical
energy, such as ATP, sufficient to permit a selected motor protein to
generate mechanical force and thus move along a microtubule track. An
effective amount can easily be determined by a person skilled in the art
without undue experimentation.
As used herein, "ATP" means adenosine triphosphate, a mononucleotide that
stores chemical energy that is used by motor proteins, such as kinesin,
for producing movement.
Eukaryotic cells contain thousands of components that are sorted and
distributed through specific bio-recognition and directed active
transport. Numerous cellular components are synthesized, processed, and
utilized in distinct cellular locations, often undergoing additional
processing during transit. Families of motor proteins, which transduce
chemical energy released by ATP hydrolysis into mechanical force and
motion, haul these cellular components along tracks of actin or
microtubule filaments to specific locations. Individual motor proteins are
hitched to their specific cargo through unique recognition domains, which
specify their cellular function. The specific function of kinesin is to
recognize and transport a subset of neuronal vesicles from the cell body
to axonal synapses. W. M. Saxton et al., Kinesin Heavy Chain Is Essential
for Viability and Neuromuscular Functions in Drosophila, but Mutants Show
No Defects in Mitosis, 64 Cell 1093 (1991). The present invention mimics
the separation functions of kinesin in nerve cells, as will become clear
from the following description.
FIG. 1 shows a diagrammatic representation of a nerve cell 4 or neuron
comprising a cell body 8, containing a nucleus 12 and neuronal vesicles
16, 20, and 24; an axon 28, containing microtubules 32; and a synapse 34.
Kinesin molecules 36 bind to a subset of neuronal vesicles 40 (FIG. 2) and
transport them on microtubules through the axon. Vesicle transport can
occur over distances up to a meter.
FIGS. 3-5 depict an illustrative micromachined separation device according
to the present invention. FIG. 3 shows a schematic diagram of a
micro-fabricated device that exploits a motor protein, such as kinesin,
and immobilized microtubules for recognizing, separating, and detecting a
selected molecule on a single silicon chip. The device 44 comprises a
loading reservoir 48 joined to a receiving reservoir 52 by a channel 56
containing immobilized microtubules. Advantageously, access ports or holes
60 are provided in the loading reservoir and receiving reservoir to permit
loading of the loading reservoir and removal of separated molecules from
the receiving reservoir. Microtubules are aligned and immobilized in the
channel such that the long axes of the microtubules are substantially
parallel to the long axis of the channel.
FIG. 4 shows an illustrative embodiment of such a micro-fabricated device.
The device 64 comprises a substrate 68 into which are micromachined a
loading reservoir, a channel, and a receiving reservoir (as best shown in
FIG. 3). A coverslip or cover plate 70 is bonded to the substrate 68 to
enclose the loading reservoir, channel and receiving reservoir, as will be
explained in more detail momentarily. Pipet tips 72 are preferably coupled
to access holes (illustrated in FIG. 3) formed in the cover plate 70 to
permit access to the loading reservoir and receiving reservoir. It will be
appreciated that access ports could be provide in other designs, such as
through micromachining in the substrate. FIG. 5 shows a cross section
through a channel 76 formed in a substrate 80 and covered or enclosed by a
cover plate 84. By "enclosed" is meant that the cover plate is placed over
the loading reservoir, channel, and receiving reservoir, and is preferably
bonded to the substrate such that liquid placed in the loading reservoir,
channel, or receiving reservoir does not leak out and such that the
coverslip does not move with respect to the substrate and thus disturb the
contents of the device. Thus, it is intended that the loading reservoir,
channel, and receiving reservoir are in liquid communication, but that the
liquid does not leak from the loading reservoir or channel into the
receiving reservoir, or vice versa. The access holes permit loading and
removal of solutions in the device.
Suitable materials for the substrate and cover plate include glass,
silicon, silica, and the like. Any other material that would be functional
for undergoing the micromachining process and would be compatible with
immobilizing microtubules, the motor-ligand composition, ATP, the selected
molecules to be separated, and a detection system that may be employed
would also be suitable.
In another embodiment of the present invention, a detection system is
coupled to the separation system previously described for monitoring the
progress of separating a selected molecule from a mixture of molecules.
FIG. 8 shows a schematic representation of such a detection system 100.
There is shown a micromachined device 104 to which is coupled a standard
epifluorescence microscope. An argon ion laser 108 emits a laser beam 112
(488 nm) that is reflected by a dichroic beam splitter 116 such that the
beam passes through an objective lens 120 (Zeiss 63+, 1.4 NA) onto the
microchannel of the separation device 104. A fluorescent intercalating
dye, such as YOYO-1 (Molecular Probes, Eugene, Oreg.), with an excitation
maximum of 491 nm and an emission maximum of 509 nm is suitable for
detecting separation of DNA. The focused laser beam contacts the dye to
excite fluorescence from transported DNA molecules. The fluorescence 124
is collected by the objective lens 120 and focused, and then passed
through a bandpass filter 128, and onto a slit in the front of a
photomultiplier tube 132. The photomultiplier tube produces a signal that
is transmitted to a PC-based data acquisition system 136 (Labview,
National Instruments, Inc.) for processing, quantitation, a storage.
One of the characteristics of kinesin that makes it particularly
well-suited for application in a separation device is that it remains
associated with the microtubule surface through thousands of ATP
hydrolysis and motility cycles. J. Howard et al., Movement of Microtubules
by Single Kinesin Molecules, 342 Nature 154 (1989). This means that a
single kinesin molecule will move many micrometers, often completely to
the end of a microtubule, without dissociating from its microtubule track.
This property is likely due to cooperativity between the two motor domains
of kinesin heavy chain dimers that results in one or the other of the
motor domains being tightly bound at all times. D. D. Hackney, Evidence
for Alternating Head Catalysis by Kinesin During Microtubule-stimulated
ATP Hydrolysis, 91 Proc. Nat'l Acad. Sci. USA 6865 (1994). Myosin and
dynein do not exhibit this property, but dissociate from their tracks
between cycles. Microtubules are polar filaments because they are
assembled from asymmetric tubulin subunits. The asymmetry is recognized by
kinesin, which moves only toward what is referred to as the plus-end of
the microtubules. Another member of the kinesin family, ncd, moves toward
the minus-end of the microtubules.
As reviewed briefly above, the kinesin heavy chain can be divided into
three domains: the motor domain (amino acid residues 1-340), the
coiled-coil stalk (amino acid residues 341-800), and the tail domain
(amino acid residues 801-975). The motor domain of Drosophila kinesin
contains 5 cysteine residues. Apparently, these cysteine residues are not
critical to kinesin activity since kinesin motility is not sensitive to
treatment with N-ethyl maleimide. Therefore, it is possible to chemically
couple a probe, such as an oligonucleotide, to cysteine residues in the
kinesin stalk without disrupting kinesin motor domain function. The
relevant region (amino acid residues 340-595) of the Drosphila kinesin
stalk contains only one cysteine residue, at position 441. In initial
examples of the operation of the present invention, oligonucleotide
ligands are coupled to this cysteine residue. In other embodiments, a
modified kinesin molecule has been constructed wherein the stalk is
truncated at residue 410 and a cysteine residue is coupled thereto. In
practice, the length of the kinesin molecule is limited only by
functionality. Generally, however, it is advantageous to limit the size of
the kinesin molecule to about 410-900 amino acid residues per chain
because expression and manipulation of proteins is generally easier with
smaller proteins as opposed to larger proteins. SEQ ID NO:2 contains the
nucleotide sequence of the Drosophila kinesin gene from kinesin cDNA
including the 5' untranslated region, the complete coding region up, and
the 3' untranslated region. This sequence of the entire gene is set forth
in J. T. Yang et al., A Three-domain Structure of Kinesin Heavy Chain
Revealed by DNA Sequence and Microtubule Binding Analyses, 56 Cell 879-89
(1989), hereby incorporated by reference.
Methods have been developed for manipulation of the microtubule component
of the active separation device. Microtubules can be routinely reassembled
in vitro from tubulin purified from bovine brains. The nucleation,
assembly, and disassembly reactions of microtubules have been well
characterized over the last 20 years. L. U. Cassimeris et al., Dynamic
instability of microtubules, 7 Bioessays 149 (1988).
EXAMPLE 1
In this example, standard cross-linking chemistry is used to covalently
attach an oligonucleotide to the carboxy-terminus of a genetically
truncated kinesin protein. Oligonucleotides can be synthesized with
modified nucleotides that contain either a thiol or an amino group for
crosslinking to the truncated kinesin protein. Oligonucleotides are
synthesized according to methods well known in the art, such as S. A.
Narang et al., 68 Meth. Enzymol. 90 (1979); E. L. Brown et al., 68 Meth.
Enzymol. 109 (1979); U.S. Pat. Nos. 4,356,270; 4,458,066; 4,416,988;
4,293,652, which are hereby incorporated by reference.
The kinesin motor protein used in this example is a 441 amino acid residue
genetically truncated version with an additional 6 histidine residues
coupled to the C-terminal Cys residue to aid in purification (SEQ ID
NO:3). This kinesin protein is expressed in E. coli according to methods
well known in the art. This kinesin motor protein can be made by digesting
pET-K447, described in J. G. Yang et al., Evidence That the Head of
Kinesin Is Sufficient for Force Generation and Motility in Vitro, 249
Science 42-47 (1990), with PvuII, and then digesting with exonuclease,
polishing the ends, and religating to obtain a plasmid that encodes the
441 amino acid residue kinesin. Expression of the protein is obtained by
transforming E. coli strain BL21 (DE3), A. H. Rosenberg et al., 56 Gene
125 (1987), growing overnight cultures of the transformed bacteria,
diluting the overnight culture 1:100 in LB medium, J. Miller, Experiments
in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor,
N.Y. (1972), supplemented with ampicillin and shaking at 37.degree. C. for
2 hours. The culture is then made 0.1 mM with IPTG, and shaken at
22.degree. C. for 10 hours. The cells are then lysed for protein
preparation as follows. The cells are harvested by centrifugation (SORVALL
GSA rotor; 8000 rpm, 5 minutes, 4.degree. C.). The pellet is resuspended
in lysis buffer (0.1 M PIPES, pH 6.9, 1 mM MgCl.sub.2, 1 mM EGTA, 2 mM
dithiothreitol) and centrifuged in a weighed tube (5 minutes, 8000 rpm,
SORVALL SS34 rotor, 4.degree. C.). The pellet is weighed and resuspended
in lysis buffer supplemented with the protease inhibitor
phenylmethylsulfonyl fluoride (PMSF) at 1 mM. Each gram of cells is
resuspended in 4 ml of buffer. The resuspended cells are lysed by
sonication. The lysed cells are released into a tube sitting on ice, and
centrifuged (SS34 rotor, 10,000 rpm, 30 minutes, 4.degree. C.). The
supernatant is referred to as the cell extract.
In one illustrative method, the kinesin heavy chain protein is enriched by
microtubule affinity as follows. The cell extract is mixed with
microtubules, prepared according to the procedure of Example 4, incubated
at room temperature for 15 minutes, and centrifuged through a 2-ml sucrose
cushion (15% sucrose, 20 .mu.M taxol, 1 mM GTP in lysis buffer with 1 mM
PMSF) in a swinging bucket rotor (54,000 g, 35 minutes, 22.degree. C.).
The pellet is resuspended in lysis buffer supplemented with protease
inhibitor, taxol, and GTP, and centrifuged at 100,000 g. The kinesin heavy
chain protein is released from microtubules by resuspending the pellet
(from 1 ml of cell extract) in 100 .mu.l of lysis buffer containing 10 mM
ATP, 10 mM MgSO.sub.4, and 0.1 M KCl, incubating at room temperature for
15 minutes, and centrifuging at 100,000 g for 30 minutes at 22.degree. C.
The supernatant containing enriched kinesin protein is divided into
portions, frozen with liquid nitrogen, and stored at -70.degree. C.
An alternative illustrative method of enrichment is by ammonium sulfate
precipitation. The kinesin heavy chain protein is precipitated in a
saturated ammonium sulfate solution (supplemented with 10 mM EDTA,
adjusted to pH 8.2 with NH.sub.4 OH, and stored at 4.degree. C.), which is
added dropwise with constant stirring until the final concentration of
ammonium sulfate is 35%. This concentration gives the best enrichment of
kinesin heavy chain protein relative to other bacterial proteins. The
mixture is stirred in the cold for 30 minutes, and centrifuged (SS34
rotor) at 10,000 rpm for 15 minutes. The pellet is resuspended in lysis
buffer with protease inhibitors (200 .mu.l of buffer for 10 ml of cell
extract), and dialyzed in 1 liter of lysis buffer for 6 hours with one
change. The dialyzed sample is clarified by centrifugation at 150 | | |
