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MOLECULAR MOTOR
FIELD OF THE INVENTION
This invention relates to molecular motors, and particularly such motors
that are powered by proteins.
BACKGROUND OF THE INVENTION
One of the [FUNDAMENTAL] properties of biological organisms is the ability
to move, or to at least transport cellular components, even on a molecular scale. The
biological structure that permits macroscopic movement in animals is muscle, which
can be either striated (skeletal), smooth, or cardiac. The molecular structure and
function of muscle has been the subject of scientific fascination and research for
over a century. As early as the [1840S,] William Bowman had suggested that
striations in skeletal muscle represented bands of intracellular material with differing
refractive indices. These intracellular materials were eventually identified as actin
and myosin.
The contractile unit in skeletal muscle is known as a myofibril, which
consists of a series of Z-disks to which are attached thin filaments of actin. The Z-
disks divide each myofibril into repeating units called sarcomeres, and within each
sarcomere is a thick filament of myosin which has heads that can form crossbridges
to the actin. In the presence of ATP, the myosin heads undergo a conformational
change that causes the cross bridges to link to the actin, and the myosin heads move
the actin filaments relative to the myosin filaments. This movement brings the Z-
disks closer together, which on a macroscopic level contracts (shortens) the muscle,
and implements musculoskeletal function. Although cardiac and smooth muscle
differ in their cellular architecture from skeletal muscle, they too rely on the
interaction of myosin and actin to contract.
The myosin molecule consists of six polypeptide subunits: two identical
heavy chains with a molecular weight of about 200,000 kDa each, and four light
chains of about 20 kDa each. In electron micrographs, purified myosin looks like a
long thin rod containing two globular heads protruding at one end. This two-headed
type of myosin is called myosin II to distinguish it from the smaller, single headed
myosin I molecule (having a shorter tail) that is involved in cytoplasmic movements
in some [NONMUSCLE] cells. The functions of portions of the myosin molecule have
been investigated by using the protease trypsin to cleave the myosin II molecule into
two fragments called light meromyosin (a coiled tail portion) and heavy meromysin
(which contains the globular heads of the molecule, and a portion of the coiled tail).
The function of actin and myosin, and their molecular structure, are more fully
described in Kendrew, The Encyclopedia of Molecular Biology, 1994, pages 688-
[691;] and Kleinsmith and Kish, Principles of Cell and Molecular Biology, second
edition, 1995, chapter 13, which are incorporated by reference.
A variety of motor proteins other than actin and myosin are also known.
The motor protein kinesin, for example, was discovered in 1985 in squid axoplasm.
Vale et al., [CELL 42:] 39-50,1985. Kinesin is just one member of a very large family
of motor proteins. Endow, Trends Biochem. Sci. 16: 221-225,1991; Goldstein,
[TRENDS CELL BIOL.] 1: 93,1991; Stewart et al., Proc. Natl. [ACAD.] Sci. USA 88: 8470-
8474,1991. [ANOTHERSUCHMOTORPROTEINISDYNEIN. LIETAL., J. CELLBIOL.] 126: 1475-
1493,1994. Kinesin, [DYNEIN,] and related proteins move along microtubules,
whereas myosin moves along actin filaments. Like myosin, kinesin is activated by
ATP.
Kinesin is composed of two heavy chains (each about 120 kDa) and two
light chains (each about 60 kDa). The kinesin heavy chains include 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 a-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.
Purified motor proteins are capable of generating movement even outside
biological organisms. The motility activity of purified kinesin on microtubules has,
for example, been demonstrated in vitro. Vale al., [CELL 42:] 39-50,1985. Full-length
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.
Yang et [AL.,] Science 249: 42-47,1990; Stewart et al., Proc. Natl. Acad. Sci. USA
90: 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 (Yang et al.), glutathione S-transferase (Stewart et al.), and biotin carboxyl
carrier protein (Berliner, 269 J. [BIOL. CHEM.] 269: 8610-8615,1994).
Similarly, methods have been developed for purification or recombinant
production and manipulation of motor proteins, and methods of attaching actin to
non-biological substrates are also known. Ishima et al., [CELL 92:] 161-171,1998.
[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. Cassimeris et al., Bioessays 7 : 149-154,1987. More
recently, recombinant tubulin has been produced in yeast. Davis et al., Biochemistry
32: 8823-8835,1993.
Efforts have been made in the past to harness the molecular activity of
motor proteins for useful work outside of biological organisms. U. S. Patent No.
5,830,659, for example, disclosed a system for purifying a molecule of interest from
a mixture by aligning microtubules in a separation channel leading out of a liquid
reservoir. A kinesin-ligand complex was then added to the liquid reservoir, in the
presence of ATP, and the ligand was selected to bind to the molecule of interest in
the liquid. When the kinesin came into contact with the microtubules in the channel,
the kinesin-ligand (and its bound molecule of interest) were transported through the
channel into a collection reservoir, so that the molecule of interest was purified away
from the mixture.
Another motor protein device is shown in Japanese patent 5-44298 (JP 5-
44298), which describes a pump for moving liquid. Actin is mounted onto a surface
of a container in the direction of the desired flow, and meromyosin and ATP are
supplied in the liquid. The interaction of the meromyosin and actin"push"the
liquid in the direction of flow.
Nicolau et [AL., SPIE] 3241: 36-46,1997 discusses constructing a molecular
motor or engine using actin and myosin. A rotatable gear is mounted on a stationary
base, and the gear has teeth to which arms of actin are attached. Using lithographic
techniques of the type used in semiconductor fabrication, a track of myosin is laid
down along the peripheral edge of the stationary base so that the arms of actin on the
rotatable gear can adhere to the track, and pull the teeth of the gear along the myosin
track when ATP is supplied to the system. This arrangement is apparently designed
to rotate the gear, and impart rotation to a driven gear that engages the driving gear.
However, the myosin track in such a device would be crushed by the teeth of the
gear as the gear rotates, or would jam.
Moreover, precise microlithographic positioning of the actin and myosin
molecules would be difficult, and perhaps unfeasible, and alignment of the actin
arms along the myosin track could not be maintained. It also does not appear that
the molecular motor could be scaled up to macroscopic proportions, nor is it clear
how the power or speed of the device could be controlled.
It is a goal of certain embodiments of the present invention to solve some
of the problems of prior approaches by devising a molecular motor that is more
easily fabricated, and may if desired be scaled up to macroscopic proportions.
It is also a goal of some embodiments to devise such a molecular motor
in which power and speed of the motor can be more conveniently controlled.
SUMMARY OF THE INVENTION
The molecular motor of the present invention includes first and second
complementary two dimensional arrays of a motor protein, for example adhered to a
substrate surface. The first and second arrays of motor proteins are in sufficiently
close contact to interact and directionally move one array (and its attached substrate)
relative to the other. This action in turn moves a driven member, such as a shaft or
gear, to convert the movement into useful power that can produce work.
In some embodiments, there are multiple layers of nested (for example
concentric) complementary first and second arrays that interact with one another to
directionally move the first and second arrays relative to one another. The arrays
may be adhered to a curved surface, such as a continuous curved surface of rotation
having a longitudinal axis and an internal radius (for example a cylinder or cone).
Multiple concentric cylinders or nested cones (which rotate around a common
central longitudinal axis) can form a series of complementary surfaces to which the
arrays are adhered,
In particular embodiments, the motor proteins are actin and myosin, and
the motor includes a source of ATP for activating the myosin to operate the motor.
The ATP can be supplied in a liquid that flows longitudinally through the rotatable
surfaces on which the arrays are adhered, or the ATP containing liquid may be
infused through perforations in surfaces on which the arrays are disposed, to allow
permeation of an ATP containing liquid through the surfaces to the motor proteins.
An array of the first motor protein may be coated on a first curved
surface, and an array of the second motor protein may be coated on a second
complementary curved surface, such that the first and second motor proteins interact
to move the second surface in a predetermined direction relative to the first surface.
In this example, one of the arrays is coated on an outer surface of a cylinder, shaft or
cone, and another of the arrays is coated on an inner surface of a surrounding
structure having a complementary shape that substantially conforms to a shape of the
outer surface of the cylinder, shaft or cone. The directional movement of the second
surface moves a driver, such as an internal shaft or cylinder in the motor.
Alternatively, the driver may be an outer curved surface of the motor (such as an
outer surface of an outermost cylinder of the motor). The driven member can take a
variety of forms, such as a rotating shaft, a propeller, a wheel, a lever-arm, a gear
system, or a pulley system.
An advantage of the disclosed motor is that the arrays can be of a
preselected dimension that provides a preselected power output of the motor. For
example, the length of a cylinder on which the complementary arrays are coated can
be selected to vary the power output. Alternatively, a speed of rotation of the motor
can be varied by preselecting the number of multiple nested complementary arrays.
Alternatively, the speed of rotation can be controlled by altering the concentration of
ATP to which the motor proteins are exposed. As the concentration of ATP
increases, the speed of the motor will increase up to a maximum speed, at which all
the motor proteins are maximally functioning.
In a more specific embodiment, the molecular motor includes a series of
concentric tubes or hollow cones, wherein each of the tubes or hollow cones has an
outer surface and an inner surface. A first motor protein array (such as an actin
array) is attached in a continuous ring of a selected width around the outer surface of
each of the tubes or cones, and a second motor protein (such as myosin) is attached
in a continuous complementary array of a corresponding width around the inner
surface of each of the tubes or cones. When actin/myosin are the motor proteins, the
actin is applied directionally to the surfaces, and the inner and outer surfaces are in
sufficiently close contact that the motor proteins interact to move the tubes or cones
relative to one another, in a direction determined by the directional application of the
actin to its surface.
The motor proteins can be attached to the surfaces in a variety of ways.
The actin, for example, can be expressed by recombinant techniques as a fusion
protein with a histidine tag, which is then attached to a nickel-coated surface.
Alternatively, the actin can be expressed with an S-tag which binds to an S-protein
coated surface, or with a streptavidin tag which binds to biotin on a substrate
surface. In another specific, non-limiting example, gelsolin is used to attach the
actin to a surface (e. g. see Suzuki et [AL.,] Biophys. J. 70: 401-408,1996).
In particular embodiments, the first motor protein (for example actin) is
directionally attached on the outer surface of a rotatable cylinder or cone in an array
that extends both longitudinally along and circumferentially around the tube or cone,
and the second motor protein (such as myosin) extends both longitudinally along
and circumferentially around the tube or cone in a complementary array of similar
size.
The invention also includes a method of making a molecular motor, by
providing a first continuous curved surface which rotates around a longitudinal axis,
and a second curved surface which rotates around the longitudinal axis, and is
complementary in shape to the first surface. A first motor protein (such as actin) is
directionally adhered to the first surface, and a second motor protein (such as
myosin) is adhered to the second surface, such that the first and second motor
proteins interact to move the first and second surfaces relative to one another. In
particular embodiments, the actin is adhered to the surface with a tag (for example a
recombinantly expressed tag such as histidine, an S-tag or streptavidin) that interacts
with a component of the first surface. The actin may be directionally applied to the
first curved surfaces by rotating the curved surface in an actin containing solution.
In certain embodiments, the motor proteins can be portions of actin and
myosin that are able to function to move the surfaces relative to one another. For
example, heavy meromyosin or myosin I can be used instead of myosin II. In other
embodiments, the motor proteins are microtubules and kinesin, or [FUNCTIONAL]
fragments thereof that are sufficient to move the surfaces. The kinesin can be, for
example, the N-terminal [410 AMINO] acid residues of kinesin.
The motor of the present invention may be a micromachined device
constructed on a micrometer-scale, but the motor can also be constructed on a much
larger scale by coating larger surfaces with the motor proteins, which can be purified
from biological tissues (such as muscle) or produced in large quantities using
recombinant techniques.
The molecular motors of the present invention are believed to operate
much more efficiently than conventional engines that use large temperature
differentials or magnetic fields to create rotary motion with energetic efficiencies
less than about 35%. The Carnot efficiency of an internal combustion engine is
56%, but other losses reduce the efficiency to about 25%. Many such engines also
depend on fossil fuels that create air pollution and may induce global warming as a
consequence of the combustion of such fuels.
Muscles use contractile or motor molecules to create macroscopic motion
with efficiencies near 70%, and the molecular motors of the present invention can
use similarly efficient systems to create useful energy. This can be accomplished
while producing substantially no pollution, because sugar (or ATP [ITSELF)] could be
used to fuel the motors, and the waste products (ADP and Pi) are biologically useful
or biodegradable. In addition, the isothermal conditions under which the motor
operates imply low materials stress, and easier construction and maintenance.
The biologically compatible nature of these devices also makes them
suitable for medical applications. Biologically based engines can use sugar in the
blood (via substrate level phosphorylation glycolysis) as fuel, to replace
neuromuscular function lost to diseases such as myasthenia gravis or muscular
dystrophy. Alternatively, the motor can be used to perform the mechanical functions
of a prosthetic implant.
The foregoing and other objects, features, and advantages of the
invention will become more apparent from the following detailed description of
several embodiments which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG. [1] is a schematic illustration of one embodiment of the molecular
motor, in which actin is directionally applied on an outer surface of a solid internal
cylinder, myosin is coated on an internal surface of a surrounding complementary
hollow cylinder, and rotation of the internal cylinder drives a rotary gear. Portions
of the outer cylinder are broken away to illustrate that the arrays of actin and myosin
extend along the length of the device.
FIG. 2 is a schematic illustration similar to FIG. [1,] but wherein the
surfaces are on cones instead of cylinders.
FIG. 3A is a side elevational and FIG. 3B is a cross sectional schematic
end view of an alternative embodiment of the invention in which the layer of actin
surrounds the myosin layer, the inner cylinder is fixed to a stationary bracket, and
rotation of the outer cylinder rotates a propeller.
FIGS. 4A through 4D are successive schematic views illustrating a
conventional view of the interaction of actin and a single myosin head, to
demonstrate how an actin coated surface is moved by the myosin.
FIGS. SA through 5E are schematic end views of cylinders similar to
those shown in FIG. [1,] showing a subset of myosin heads that change conformation
substantially in concert to move the internal actin coated cylinder of the motor.
Other myosin heads (not shown) are randomly moving through different stages of
the conformational changes, without necessarily moving in concert, but only a single
subset of myosin heads have been shown for purposes of explanation.
FIG. 6 is a schematic side view of an alternative embodiment of the
motor having multiple, nested, concentric complementary cylinders on which the
actin and myosin are coated.
FIG. 7A is a schematic end perspective view of two interengaging
complementary cylinders that can be interengaged to assemble a molecular motor of
the present invention.
FIG. 7B is a side view of the complementary cylinders of FIG. 4,
illustrating the differing outer diameters of the two cylinders.
FIG. 8 is a schematic illustration of one embodiment of the molecular
motor, in which ATP is supplied from a reservoir. Separate feed lines are used to
supply the ATP to the motor. Each feed line [(ATP,, ATP2,] and ATP3) has a control
switch or valve (designated"X"on the [ATP,, ATP2,] and ATP3 feed lines). In one
embodiment, the control valves are separately controlled.
FIG. 9 is a schematic illustration of another embodiment of the molecular
motor, which includes separate units in series. In this embodiment, segments of a
molecular motor, separated by impermeable barriers, are connected in series by a
shaft. The barrier is designed to prevent diffusion between the molecular motor
units. In this embodiment, ATP is supplied from a reservoir through separate feed
lines (designated [ATP,, ATP2, ATP3] and [ATP4).] Each feed line (ATP,, ATP2, ATP3
and ATP4) has a separately controlled switch or valve (designated"X"on [ATP,,]
[ATP2, ATP3] and ATP4 feed lines).
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Definitions
The following definitions and methods are provided to better define the
present invention and to guide those of ordinary skill in the art in the practice of the
present invention. Definitions of common terms may also be found in Rieger et [AL.,]
Glossary [OF GENETICS. CLASSICAL AND MOLECULAR, 5TH EDITION,] Springer-Verlag: New
York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. The
standard one and three letter nomenclature for amino acid residues is used (such as
H or His for Histidine).
Additional definitions of terms commonly used in molecular genetics can
be found in Benjamin Lewin, Genes V published by Oxford University Press, 1994
(ISBN 0-19-854287-9); Kendrew et al (eds.), The Encyclopedia ofMolecular
[BIOLOGY,] published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and
Robert A. Meyers (ed.), Molecular Biology and [BIOTECHNOLOGY. A COMPREHENSIVE]
Desk Reference, published by VCH Publishers. [INC.,] 1995 (ISBN 1-56081-569-8).
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 motor comprising"a cylinder"includes
a system containing one or more cylinders, and reference to"a motor protein"
includes reference to one or more motor proteins.
Micromachining, micromachined, and similar terms refer to the
processes used to create micrometer-sized structures with primarily mechanical
functions on substrates such as glass, silicon, silica, or a photoreactive polymer-
coated chip.
Motor protein means a protein that transduces chemical energy into
mechanical force and motion. Such motor proteins often exist in complementary
pairs, such as actin and myosin, or kinesin and microtubules. Particular disclosed
motor proteins are actin/myosin and kinesin/microtubles. The motor proteins can be
used in any form that is capable of transducing the chemical energy (such as the
energy of ATP) into mechanical force and motion. Hence variants or fragments of
the molecules can be used, such as myosin I or myosin [II,] or heavy meromyosin
(although light meromyosin would not be suitable because it lacks the heads which
change conformation to transduce the chemical energy). Similarly, variant or
mutant forms of the motor proteins, such as variant actin or myosin (for example
proteins in which conservative amino acid substitutions have been made) are also
included, as long as they retain the motor activity. Actin is a directionally oriented
molecule, that (when applied directionally to a substrate) helps direct myosin along a
substrate in a direction determined by the orientation of the actin molecules on the
surface. Actin and myosin have been well studied, and mutations that affect their
function have been reported in the scientific literature to provide guidance about
making mutants. See, for example, J. Cell. Biol., 134: 895-909,1996; J. Biol. Chem.
269: 18773-18780,1994; and Bioessays 19: 561-569,1997.
The motor proteins may also include kinesin and related proteins, such as
ncd, as disclosed in Endow et al., Nature 345: [81-83,1990,] that are highly
processive, i. e. which do not readily detach from directional 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. Kinesin moves
toward the plus-end of microtubules, whereas ncd moves toward the minus-end of
microtubules. Hence, like actin, the microtubules can be applied directionally to a
substrate to pre-select a direction of rotation of the surfaces relative to one another.
The direction of rotation can be varied, depending on the complementary motor
protein which is selected (for example, kinesin or ncd).
The motor proteins also include species variations, and various sequence
polymorphisms that exist, wherein amino acid substitutions in the protein sequence
do not affect the essential functions of the protein.
Coupling of a motor protein to the surfaces of the rotatable cylinders or
cones of the motor can be accomplished by any method known in the art, as long as
the motor activity of the protein is preserved. An example of a method of expressing
actin as a fusion protein that is then coupled to a substrate is given in Example 4, in
which a fusion protein is expressed by recombinant DNA technology. Briefly, a
gene encoding a motor protein is operably linked to a gene encoding a selected tag
(such as poly-His or streptavidin) to construct a gene fusion, which is then expressed
in a suitable expression system such as E. coli or yeast to produce the fusion protein.
Coupling of the motor protein to the substrate can also be accomplished by other
methods, such as chemical coupling or purified proteins.
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.
ATP means adenosine triphosphate, a mononucleotide that stores
chemical energy that is used by motor proteins, such as myosin and kinesin, for
producing movement. ADP refers to adenosine diphosphosphate.
GTP means guanosine 5'-tripospahte, a mononucleotide that stores
chemical energy.
[CDNA] (complementary DNA): a piece of DNA lacking internal, non-
coding segments (introns) and regulatory sequences which determine transcription.
[CDNA] is synthesized in the laboratory by reverse transcription from messenger RNA
extracted from cells.
Deletion: the removal of a sequence of DNA, the regions on either side
being joined together.
Fuel source means a molecule that stores chemical energy. In one
embodiment, the energy molecule is a nucleoside triphosphate (NTP), such as ATP
or GTP.
Motor protein gene: a gene (DNA sequence) encoding a motor protein
(such as actin or myosin). A mutation of the gene (to produce variant forms of the
motor protein) may include nucleotide sequence changes, additions or deletions.
The term"gene"is understood to include the various sequence polymorphisms and
allelic variations that exist within the population. This term relates primarily to an
isolated coding sequence, but can also include some or all of the flanking regulatory
elements and/or intron sequences.
NTP means a nucleoside 5'-triphosphate, e. g. ATP or GTP.
Isolated: requires that the material be removed from its original
environment. For example, a naturally occurring DNA or protein molecule present
in a living animal is not isolated, but the same DNA or protein molecule, separated
from some or all of the coexisting materials in the natural system, is isolated.
Operably linked: A first nucleic acid sequence is operably linked with a
second nucleic acid sequence when the first nucleic acid sequence is placed in a
functional relationship with the second nucleic acid sequence. For instance, a
promoter is operably linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally, operably linked DNA
sequences are contiguous and, where necessary to join two protein coding regions, in
the same reading frame.
ORF: open reading frame. Contains a series of nucleotide triplets
(codons) coding for amino acids without any termination codons. These sequences
are usually translatable into protein.
PCR: polymerase chain reaction. Describes a technique in which cycles
of denaturation, annealing with primer, and then extension with DNA polymerase
are used to amplify the number of copies of a target DNA sequence.
Purified: the term"purified"does not require absolute purity; rather, it
is intended as a relative term. Thus, for example, a purified protein preparation is
one in which the protein referred to is more pure than the protein in its natural
environment within a cell. The term"substantially pure"refers to a purified protein
having a purity of at least about 75%, for example 85%, 95% or 98%.
Recombinant: A recombinant nucleic acid is one that has a sequence that
is not naturally occurring or has a sequence that is made by an artificial combination
of two otherwise separated segments of sequence. This artificial combination is often
accomplished by chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e. g., by genetic engineering
techniques.
Sequence identity: the similarity between two nucleic acid sequences, or
two amino acid sequences, is expressed in terms of the similarity between the
sequences, otherwise referred to as sequence identity. Sequence identity is frequently
measured in terms of percentage identity (or similarity or homology); the higher the
percentage, the more similar are the two sequences.
Methods of alignment of sequences for comparison are well-known in the
art. Various programs and alignment algorithms are described in: Smith and
Waterman, Adv. [APPL.] Math. 2: Needleman and Wunsch, J. [MOL. BIO.]
48: 443,1970; Pearson and [LIPMAN, METHODS IN MOL. BIOL.] 24: 307-31,1988; Higgins
and Sharp, Gene 73: 237-44,1988; Higgins and Sharp, [CABIOS 5:] [151-3,1989;] Corpet
[ET AL., NUC. ACIDS RES.] 16: 10881-90,1988; [HUANGETAL., COMP. APPL. BIOSCI.] 8: 155-
65,1992; and Pearson et al., Meth. Mol. Biol. 24: 307-31,1994.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.
Mol. Biol. 215: 403-10,1990) is available from several sources, including the National
Center for Biological Information (NBCI, Bethesda, MD) and on the Internet, for use
in connection with the sequence analysis programs [BLASTP,] blastn, [BLASTX,] tblastn and
tblastx. It can be accessed at http://www. ncbi. nlm. nih. [GOV/BLAST/.] A description of
how to determine sequence identity using this program is available at
http://www. ncbi. nlm. nih. [GOV/BLAST/BLAST_HELP.] html.
Variants or homologs of the motor protein are typically characterized by
possession of at least 70% sequence identity counted over the full length alignment
with the disclosed amino acid sequence using the NCBI Blast 2.0, gapped blastp set to
default parameters. Such homologous peptides will more preferably possess at least
75%, more preferably at least 80% and still more preferably at least 90%, 95% or 98%
sequence identity determined by this method. Sequence identity can be determined, in
one instance, by aligning sequences and determining how many differences there are
in the aligned sequence, and expressing these differences as a percentage. When less
than the entire sequence is being compared for sequence identity, homologs will
possess at least 75% and more preferably at least 85% and more preferably still at least
90%, 95% or 98% sequence identity over short windows of 10-20 amino acids.
Methods for determining sequence identity over sequence windows are described at
http://www. ncbi. nlm. nih. [GOV/BLAST/BLAST_FAQS. HTML.] For comparisons of nucleic
acid sequences of less than about 150 nucleic acids, the Blast 2 sequences function is
employed using the default 0 BLOSUM62 matrix set to default parameters, (OPEN
GAP [5,] extension gap 2). Nucleic acid sequences with even greater similarity to the
reference sequences will show increasing percentage identities when assessed by this
method, such as at least 45%, 50%, 70%, [80%,] 85%, 90%, 95% or 98% sequence
identity.
The present invention provides not only the peptide homologs that are
described above, but also nucleic acid molecules that encode such homologs.
Transformed: A transformed cell is a cell into which has been
introduced a nucleic acid molecule by molecular biology techniques. As used
herein, the term transformation encompasses all techniques by which a nucleic acid
molecule might be introduced into such a cell, including transfection with viral
vectors, transformation with plasmid vectors, and introduction of naked DNA by
electroporation, lipofection, and particle gun acceleration.
Vector: A nucleic acid molecule is introduced into a host cell, thereby
producing a transformed host cell. A vector may include nucleic acid sequences that
permit it to replicate in a host cell, such as an origin of replication. A vector may
also include one or more selectable marker genes and other genetic elements known
in the art.
Particular Embodiments
EXAMPLE 1
A particular embodiment of the molecular motor 10 is illustrated in FIG.
[1,] in which the motor is shown to include a solid inner cylinder 12 and a hollow
outer cylinder 14 that is of slightly larger diameter than inner cylinder 12. An
extension 16 of inner cylinder 12 projects from motor 10, and carries a driver in the
form of a toothed gear 18. The teeth on gear 18 mesh with the teeth of a larger gear
20, so that rotation of gear 18 in the direction of arrow 22 will rotate gear 20 in the
direction of arrow 24.
Although the dimensions of motor 10 are not critical, the inner cylinder
12 may have a diameter of 20 microns to 1 meter, for example 1 cm, while the outer
cylinder 14 may have a diameter of 40 microns to 1 meter, for example 1 cm. A
clearance distance between an outer surface of cylinder 12 and an inner surface of
cylinder 14 is, for example, in the range of 20 to 30 microns.
Referring again to FIG. [1,] a layer of actin 30 is directionally applied to
the outer surface of cylinder 12, with the directional orientation shown as arrows in
the drawing. As described in greater detail in Example [4,] the actin protein may be
expressed with a histidine tag (for example His-6) that binds to nickel. The actin is
polymerized to form actin fibers by bringing a [MG2+] concentration to physiological
levels, as described in [KORN] et al., Science [238:] [638-644,1987.] A cylinder with a
nickel outer surface is placed into the actin-His-6 fiber solution so that the fibers
attach to the surface of the cylinder. The cylinder may then be placed in a normal
(non-His-6) actin solution (for example by adding normal actin to the solution) to
extend the actin fibers to a length beyond their persistence length (at which point the
actin has no particular direction). More actin-His-6 is then added to the solution, so
that the ends of the actin cables have a His tag, and the cylinder is rotated in the
actin solution to directionally orient the actin cables, and allow the His tags at the
free end of the actin cables to attach to the nickel containing surface of the cylinder
12.
The directionality of the actin cables is schematically illustrated in FIG. [1]
by arrows 32 in the actin layer 30. As shown by the cut away portion of cylinder 14
in FIG. [1,] the coating of actin covers the curved surface of inner cylinder 12
substantially along its length, for substantially coating that surface of the cylinder.
In particular embodiments, the actin is present in a substantially continuous layer
around the circumference of the cylinder 12, for substantially the entire length of the
cylinder inside outer cylinder 14. The thickness of the actin layer may be, for
example, [1] to 10 molecules thick, and in a particular disclosed embodiment is one
molecule thick.
The myosin (for example in the form [OF MYOSIN I,] myosin [II,] or heavy
meromyosin, or variants thereof) can be adhered to the inner surface of cylinder 14,
before actin coated cylinder 12 is placed inside cylinder 14. The myosin is adhered
to the inner surface of cylinder 14 by adhesion, or by the techniques shown in Finer
et al., Nature 368: 113-119,1994, and Ishijima et al., Cell 92: 161-171,1998, as well
as Ishijima et al, Biophys. J. 70: 383-400,1996 (incorporated by reference), in which
myosin was purified and bound to a glass surface. When heavy meromyosin
(HMM) is used as the motor protein, the technique used in Suzuki et al., Biophys. J.
72: 1997-2001,1997 (incorporated by reference) can be used. In this method, HMM
(0.1 mg/ml) in an assay buffer solution (40 mM KCI, 3 mM [MGC12,2MM] EGTA,
10 mM dithiothreitol, and 20 mM HEPES at pH 7.8) is dropped on to a
polymethylmethacrylate (PMMA) substrate, and the HMM is adsorbed. PMMA is a
useful substrate, because photolithographic patterns can be made in them, if desired,
and the HMM placed into the tracks. In FIG. 1, the myosin is schematically shown
as myosin heads 34 projecting from the inner surface of cylinder 14.
Once the myosin has been adhered to the inner surface of cylinder 14,
cylinder 12 may be inserted inside cylinder 14, with both cylinders arranged
concentrically around a common longitudinal axis 36. External cylinder 14 may be
mounted to a stationary surface 38 by a bracket 40, so that the cylinder 14 remains
fixed, and inner cylinder 12 is free to rotate relative to cylinder 14, around the
central longitudinal axis 36.
In operation, a solution that contains an effective concentration of ATP is
introduced into the flow space 42 between cylinders 12,14, and allowed to flow
through the cylinders along their entire length. Particular concentrations of ATP
(Sigma Chemical Co., St. Louis, MO.) that can be supplied are solutions with an
ATP concentration of 0.1 to 1000 [U. M,] for example 1 [1M.] Greater concentrations of
ATP would activate more of the myosin molecules, and increase the speed of the
motor, by rotating cylinder 12 relative to cylinder 14. As cylinder 12 rotates,
extension 16 rotates gear 18 in the direction of arrow 22, which in turn rotates gear
20 in the direction of arrow 24. The molecular mechanism by which this rotation is
achieved is described in more detail in Example 2.
An alternative embodiment is shown in FIG. 2, which is similar to that
shown in FIG. [1,] such that like parts have been given like reference numbers plus
100. However, instead of inner and outer cylinders, the motor includes inner and
outer frusto-cones 112,114 (which for simplicity will be referred to as"cones"112,
114). FIG. 2 shows the molecular motor [110] in which the outer cone 114 is
mounted to a bracket 140 and surface [138.] Outer cone 114 is positioned around
inner cone 112, such that the cones taper in a complementary fashion, from a large
diameter base to a smaller diameter tip, and rotate around a common longitudinal
axis of rotation 136. A layer of actin 130 is directionally attached to the outer
surface of inner cone 112, while myosin 134 is adhered to the inner surface of outer
cone 114. When supplied with fuel, inner cone 112 rotates extension 116 and
driving gear 118 in the direction of arrow 122, which in turn rotates driven gear 120
in the direction or arrow 124.
An advantage of the embodiment of FIG. 2 is that the motor can be
assembled by inserting inner cone 112 inside outer cone 114, with less shearing
force than may be encountered when introducing an inner cylinder into a larger outer
cylinder. Since the smaller diameter top portion of the tapering inner cone 112 can
be introduced into the larger diameter base opening of the outer tapering cone 114,
there is a greater clearance between the inserted end and the surrounding cone than
would occur with two cylinders, each of which has a constant radius. As the inner
cone 112 is progressively inserted into the outer cone 114, the minimum desired
operational clearance between the actin and myosin layers is not reached until the
two cones reach their final operational positions. Hence the opportunity for shearing
of the actin and myosin layers, by frictional forces encountered as the motor is
assembled, is minimized.
Another alternative embodiment of the motor is shown in FIGS. 3A and
3B, in which a hollow inner cylinder 43 is surrounded by an outer cylinder 44.
Myosin 45 (with the heads shown in random states of conformational change in FIG.
3B) is coated on an external surface of inner cylinder 43, while a layer of actin 46 is
directionally applied to an inner surface of outer cylinder 44. Openings 47 are
arrayed circumferentially around outer cylinder 44, and provide passageways
through the cylinder 44 and actin layer 46, through which an ATP containing liquid
can be introduced into the space between cylinders 43 and 44. Inner cylinder 43
extends beyond an open end of outer cylinder 44, and is mounted on a stationary
bracket 48. Myosin need not be coated on the outer surface of cylinder 43 which
extends out of cylinder 44.
In operation, a liquid containing a sufficient concentration of ATP is
introduced through passageways 47, for example through manifold tubes (not
shown) which communicate with the passageways. In the presence of the ATP, the
myosin heads 45 undergo a conformational change to attach to actin layer 46 and
move it in the direction indicated by arrow 49. As the actin layer is moved, its
attached outer cylinder 44 is rotated around its longitudinal axis in the direction 49,
which in turn rotates propeller blades 51 that extend outwardly from the outer
surface of cylinder 44. The rotation of blades 51 can be converted to useful work,
such as the generation of power.
Although FIGS. 3A and 3B show perforations 47 in the external cylinder
44 for introducing liquid fuel into the motor, the liquid could similarly be introduced
into the interior of the hollow inner cylinder 43. Perforations in cylinder 43 could be
provided to direct the flow of liquid out of the inner cylinder, and this flow would be
encouraged by rotation of the surrounding outer cylinder 44.
The molecular motor can be used in a biological organism, such as a
mammal, for example to move limbs or other body parts that may have lost
neuromuscular activity. When used to move a limb, for example, the rotation of
outer cylinder 44 can be used to rotate a joint, for example to perform pronation or
supination of the forearm. In an assembly such as that shown in FIGS. 3A and 3B,
the inner cylinder can be fixed axially to a bone (such as the radius or ulna, or both),
and the rotating outer cylinder can be fixed to the humerus. Activation of the motor
would then rotate the forearm relative to the upper arm. In such an example, a motor
with multiple layers would likely be required to provide sufficient power to rotate a
joint.
In yet other applications, the molecular motor may be used in a robot, for
example to rotate joints of the extremities or trunk. Rotation of the motor can also
be used in a pump to propel fluids. Very large versions of the motor (such as
multiple cylinder embodiments about one meter wide) could also be used in
automobiles to replace conventional internal combustion motors.
EXAMPLE 2
Movement of Substrates by [CONFORMATIONAL] Change of Myosin Heads
The molecular mechanism by which conformational changes of the
myosin heads move an actin coated substrate are illustrated in FIGS. 4 and 5, which
depict a conventional version of the mechanism of muscle contraction. Although
this version is illustrated for purposes of explanation and illustration, the invention is
not limited to this theory, and covers any actual mechanism of muscle contraction
eventually discovered.
FIG. 4 shows a flat substrate 200 coated with a directionally oriented
layer of actin 202. In FIG. 4A, the myosin head 204 is shown at the end of a power
stroke which has moved substrate 200. In step 1 between FIG. 4A and FIG. 4B,
ATP binds to the myosin head 204, which causes release of the myosin head 204
from the actin 202. ATP is then rapidly hydrolyzed, leaving ADP and inorganic
phosphate [(PI)] bound to the myosin 204, and resulting in a conformational change
(FIG. 4C) in the shape of the myosin head which moves the head backward with
respect to the direction of desired movement of the actin. This change is followed
by the myosin binding to actin in a high energy state (FIG. 4D). The ADP-Pi is then
released, which results in another conformational change that moves the myosin in
the direction of arrow 206, and drives the actin filament by a distance of between 4
and 10 nm in that direction.
A similar proposed mechanism applies to the movement of a curved
substrate, such as the cylinder 212 (FIG. 5) which is coated with the layer of actin
230. FIG. 5A shows the myosin heads 234 at the end of a power stroke. Although
several myosin heads are shown in FIG. [5] undergoing uniform movements, the
myosin head which are shown are only a subset of myosin molecules that are
undergoing similar conformational changes. Although not shown in the drawing,
many other myosin molecules are simultaneously in different stages of the cycle.
In step [1,] between FIGS. 5A and [5B,] ATP binds to the myosin heads
234, which causes release of the heads from the directionally oriented actin layer
230. The ATP is subsequently hydrolyzed in step 2, leaving ADP and Pi (illustrated
as a black spot on the myosin head in FIG. [5C),] and resulting in a conformational
change that moves the myosin head in a direction opposite the direction of
movement of the directionally oriented actin. The myosin heads then attach to the
actin fibers (FIG. [5D),] and the ADP-Pi is released, resulting in a conformational
change of the myosin that drives the heads in the direction of arrow 236. This
movement in turn moves the actin in the direction of arrow 236 to turn the inner
cylinder, and power the motor.
EXAMPLE 3
Multiple Concentric Cylinders to Increase Speed of Motor
Another embodiment of the motor is shown in FIG. 6, in which multiple
concentric cylinders are used to construct a motor that can rotate at a higher speed
than a motor having only an inner and an outer cylinder. In the embodiment of FIG.
6, the motor includes a solid inner cylinder 270, an intermediate cylinder 272, and an
outer cylinder 274. Although three cylinders are shown in this example, a motor
containing many more cylinders (for example 5,10,25,50 or even more concentric
cylinders) can similarly be used.
The construction of the motor in FIG. 6 is analogous to that shown in
FIGS. 1-3, in that opposing surfaces of the cylinders are coated with complementary
pairs of motor proteins, such as actin and myosin. Hence inner cylinder 270 has a
layer of actin 276a directionally coated on its external surface, while intermediate
cylinder 272 has a coating of myosin 278a on its inner surface. Intermediate
cylinder 272 also has a directional layer of actin 276b on its outer surface, and outer
cylinder 274 has a coating of myosin 278b on its inner surface.
In operation, the outer cylinder 274 is held stationary, for example by a
bracket. When an ATP-containing liquid is introduced into the spaces between the
three cylinders, the myosin on the inner surface of outer cylinder 274 moves
intermediate cylinder 272 in the direction indicated by arrow 280. Simultaneously
the inner cylinder 270 is rotated in the direction of arrow 280 by the interaction of
the complementary actin and myosin layers on the cylinders 270,272. Hence the
rotational speed of inner cylinder 270 is the sum of the rotational speeds of
intermediate cylinder 272 and inner cylinder 270. By using even more concentric
cylinders that rotate about a common longitudinal axis, the rotational speed on the
inner cylinder can be increased correspondingly.
Alternatively, in embodiments such as that shown in FIGS. 3A and 3B in
which the outer cylinder rotates relative to a stationary inner cylinder, multiple
concentric cylinders in the motor would increase the rotational speed of the external
cylinder.
FIGS. 7A and 7B illustrate a particular mode of assembly of molecular
motors that have multiple concentric nested cylinders. A first set 281 of hollow
coaxial cylinders is held in the concentric array shown in FIG. 7A, for example by a
series of internal struts, or by affixation of an external end plate 280 (FIG. 7B) at a
closed end of the array. Set 281 includes three hollow coaxial cylinders, consisting
of an inner cylinder 282, and intermediate cylinder 284, and an outer cylinder 286.
A second set 287 of coaxial cylinders is similarly held in a concentric array by
internal struts, or affixation of an external end plate 283 at a closed end of the array
(FIG. 7B). Set 287 also includes three cylinders, consisting of an inner cylinder 288,
an intermediate cylinder 292, and an outer cylinder 290.
The overall outer diameter RI of set 281 is slightly less than an overall
outer diameter R2 of set [287,] and the corresponding arrays of the alternate sets [281,]
287 have staggered diameters from the innermost to the outermost cylinder. Hence
the outer diameter of cylinder 282 is slightly less than the inner diameter of cylinder
288. Similarly, the outer diameter of cylinder 288 is slightly less than the inner
diameter of cylinder 284, and the outer diameter of cylinder 284 is slightly less than
the inner diameter of cylinder 292.
As illustrated schematically in FIG. 7A, actin is directionally applied to
the outer surfaces of both of the cylinders 282,284 (where the directional
application of the actin is illustrated by the direction of the arrows on the outer
surfaces of those cylinders). Myosin is applied to the inner surfaces of cylinders
288,290 and 292. Hence the motor can be assembled by introducing set 281 into set
287, so that the cylinders of set 281 interdigitate with the cylinders of set 287. Once
assembled, the motor can be operated by introducing an ATP containing liquid into
the spaces between the cylinders.
In another embodiment (not shown), each actin bearing surface can have
raised circumferential ridges longitudinally spaced along it. Such raised ridges
would provide areas of reduced clearance between the inner and outer cylinders, to
increase the interaction between the cylinders.
When a set number of concentric nested cylinders is included in a
molecular motor, the motor operates at a defined maximum power and speed.
However, it may be desirable to be able to vary the power of the motor. As shown
in FIG. 8, the molecular motor may be elongated along the horizontal axis. The fuel
source (e. g., an energy molecule such as a nucleotide triphosphate, NTP) is provided
from a reservoir. In one embodiment, the fuel source ATP.
The fuel source is selected based on the enzyme system of the molecular
motor. For example, if helicases and DNA strands are included in a molecular
motor, NTPs are provided in the reservoir (see Waksman et al., Nat. Struct. Biol.
7: 20-22,2000 for a discussion of helicases). In another specific, non-limiting
example, actin and myosin are included in the molecular motor, and ATP is
provided as the fuel source in the reservoir.
The fuel source (e. g. ATP) is supplied to the molecular motor by feed
lines (designated [ATP,, ATP2, ATP3)] that are controlled by switches or valves
(designated X on feed lines [ATP,, ATP2, ATP3)] that regulate the flow rate of fuel
(e. g. ATP) through the feed lines. Power is varied by changing the amount of
available fuel along the length of the motor using the control switches or valves.
In the embodiment illustrated, there are three feed lines and switches,
however, any number of independently controlled feed lines and switches can be
utilized. The control switches or valves can be regulated individually, regulated in
groups (e. g. 2,3, or 4 valves that are regulated together), or can be regulated as a
single unit.
Thus, in one specific, non-limiting example, independently controlled
switches are utilized to control the flow of ATP through the feed lines. If three
switches and three ATP feed lines are connected to the motor, switching one of the
three independently controlled switches off decreases the power to two-thirds of the
maximal power.
Another embodiment of the molecular motor, wherein power can be
regulated, is shown in FIG. 9. In this embodiment, independent segments of a
molecular motor are provided. Each segment of the motor (shown as an independent
cylinder) is attached to one end of a feed line (designated [ATP,, ATP2, ATP3] and
[ATP4).] The other end of each feed line is connected to a fuel source (e. g. ATP) in a
reservoir, which can deliver the fuel through the feed lines to the segments of the
molecular motor. In the embodiment shown, the flow of ATP from the reservoir
through the feed lines is controlled by valves or switches (designated"X"on each
feed line). The motor segments are separated by impermeable barriers (shown
schematically as squares) that prevent, or substantially inhibit, diffusion between the
motor segments.
In the embodiment illustrated, four feed lines and switches are shown,
however, any number of independently controlled feed lines and switches can be
utilized. Moreover, each of the segments can have multiple supply lines (as in FIG.
8). The control switches or valves shown in FIG. 9 can be regulated individually,
regulated in groups (e. g. 2,3, or 4 valves that are regulated together), or can be
regulated as a single unit.
In one specific, non-limiting example, segments of the motor are
powered independently to avoid shear. For example, if the segments are numbered
sequentially, the switch can be used to prevent delivery of ATP to every other
segment (e. g. the odd segments) in order to run the molecular motor at half of the
maximum power. Similarly, the switch can be used to prevent delivery of ATP to
every third segment (e. g. those with a multiple of three) to run the molecular motor
at two-thirds power. The switch can also be used to prevent delivery of ATP to two
out of three segments to run the molecular motor at one-third power.
The segments of the motor can all be of the same length, or can have
different lengths. Altering the lengths of the segments allows variations in power.
In addition, altering the numbers of nested cylinders allows the velocity to be varied.
Thus, a range of controls is provided.
In one embodiment, a series in which the first segment has a unit length
of 1, a second segment has a unit of length 2, and third segment has a unit of length
of 4, and a fourth segment has a unit length of 8 is provided. This series can, by
binary combinations, be programmed to have from 0 to 15 units of power. One of
skill of the art will be able to determine an appropriate switching paradigm of
segments of molecular motor of various lengths such that any desired fraction of the
maximal power of the molecular motor can be achieved.
EXAMPLE 4
Preparation of Recombinant Actin
This example describes how to prepare recombinant actin molecules,
which may also contain at least one affinity tag. Such tags serve as a means by
which to attach actin to a substrate, and aid in the purification of recombinant actin.
Purified recombinant actin may be used for the molecular motor of the present
invention.
Standard molecular biology protocols are used for the expression and
purification of recombinant actin unless otherwise stated. Such methods are
described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, New York, 1989), Ausubel et al. (Current Protocols in
Molecular Biology, Greene Publishing Associates and Wiley-Intersciences, 1987),
and Innis et al., (PCR Protocols, A Guide to Methods and Applications, Innis et al.
(eds.), Academic Press, Inc., San Diego, California, 1990).
Partial or full-length [CDNA] sequences, which encode for actin, may be
ligated into bacterial expression vectors. The actin [CDNA] can be from any organism
including human, chicken or mouse, and includes wild-type, mutant, and sequence
variants thereof. In addition, the actin [CDNA] may be from any isotype of actin,
including the [A,] [P,] and y isoforms. Any sequence variants used in the present
invention will retain the ability to interact with myosin so that the myosin can move
the actin, as in muscle. Several actin [CDNA] sequences are publicly available on
GenBank at: http://www. ncbi. nlm. nih. gov/Entrez/. Examples include the human
(Accession No. J0068) and chicken (Accession Nos. V01507 J00805 K02172
K02257) a-actin genes, the mouse [P-ACTIN] gene (Accession No. X03672), and the
human y-actin gene (Accession Nos. X04098, [K00791, M24241).] It is appreciated
that for mutant or variant DNA sequences, similar systems as described below are
employed to express and produce the mutant or variant product.
DNA sequences can be manipulated with standard procedures such as
restriction enzyme digestion, fill-in with DNA polymerase, deletion by exonuclease,
extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned
DNA sequences, site-directed sequence-alteration via single-stranded bacteriophage
intermediate, or with the use of specific oligonucleotides in combination with PCR.
The host cell, which may be transfected with the vector of this invention, may be
selected from the group consisting of bacteria, yeast, fungi, plant, insect, mouse or
other animal, or human tissue cells.
The purification of recombinant fusion proteins has been made
significantly easier by the use of affinity tags that can be genetically engineered at
either the N-or C-terminus of recombinant proteins. Such tags can be attached to
actin, to aid in its purification and subsequent attachment to a substrate (see Example
1). Examples of affinity tags include histidine (His), streptavidin, S-tag, and
glutathione-S-transferase (GST). Other affinity tags known to those skilled in the art
may also be used.
In general, the affinity tags are placed at the N-or C-terminus of a
protein. Vectors containing one or multiple affinity tags are commercially available.
To prepare a Tag-actin recombinant fusion protein, vectors are constructed which
contain nucleotide sequences encoding the tag, and the actin [CDNA.] This vector
may be expressed in bacteria such as E. coli, and the protein purified. The method
of purification will depend on the affinity tag attached. Typically, the bacterial
lysate is applied to a column containing a resin having high affinity for the tag on
the fusion protein. After applying the lysate and allowing the tagged-fusion protein
to bind, unbound proteins (non-tagged) are washed away, and the fusion protein
(containing the affinity tag) is eluted.
One of the most widely used tags contains six or ten consecutive
histidine (His) residues, which has high affinity for metal ions (such as nickel ion)
which can be placed on a surface of a curved substrate to which the actin is to be
attached. A His-6 or His-10 moiety can be attached to actin using pET vectors
(Novagen, Madison, WI). The His-actin fusion protein can be purified as described
in Paborsky et al. (Anal. Biochem., 234: 60-65,1996), herein incorporated by
reference. Briefly, the cell lysate is immobilized by affinity chromatography on
[NI"-NTA-AGAROSE] (QIAGEN, Valencia, CA). After washing away unbound
proteins, for example using a buffer containing 8-50 mM imidazole, 50 mM Tris
HCI, pH 7.5,150 mM [NACI,] the bound recombinant protein is eluted using the same
buffer containing a higher concentration of imidazole, for example 100-500 mM
imidizole.
The S-tag system is based on the interaction of the 15 amino acid S-tag
peptide with the S-protein derived from pancreatic ribonuclease A. Several vectors
for generating S-tag fusion proteins, as well as kits for the purification of S-tagged
proteins, are available from Novagen (Madison, WI). For example vectors pET29a-
c and pET30a-c can be used. The S-tag-actin fusion protein may be purified by
incubating the cell [LYSTAE] with S-protein agarose, which retains S-tag-actin fusion
proteins. After washing away unbound proteins, the fusion protein is released by
incubation of the agarose beads with site-specific protease, which leaves behind the
S-tag peptide. The S-tagged protein can then be attached to the cylinder substrate,
for example by the His tag provided by this vector on the C terminus.
The affinity tag streptavidin binds with very high affinity to biotin.
Vectors for generating streptavidin-actin fusion proteins, and methods for purifying
these proteins, are described in Santo and Cantor (Biochem. Biophys. Res. Commun.
176: 571-577,1991, herein incorporated by reference). To purify the streptavidin-
actin fusion protein, the cell lysate is applied to a 2-iminobiotin agarose column
(other biotin-containing columns may be used), and after washing away unbound
proteins, the fusion protein is eluted. Biotin can be attached to the substrate (a
surface of the cylinder, such as a glass cylinder) using the techniques disclosed by
Mazzola and Fodor, Biophys. J. 68: 1653-1660,1995, which is incorporated by
reference.
The enzyme glutathione-S-transferase [(GST)] has high affinity for
glutathione. Plasmid expression vectors containing GST (pGEX) are disclosed in
U. S. Patent No. 5,654,176 to Smith, herein incorporated by reference and in
Sharrocks [(GENE,] 138: 105-8,1994, herein incorporated by reference). [PGEX] vectors
are available from Amersham Pharmacia Biotech (Piscataway, NJ). The cell lysate
is incubated with glutathione-agarose beads and after washing, the fusion protein is
eluted, for example, with 50 mM Tris-HCI (pH 8.0) containing 5 [MM] reduced
glutathione. If the GST-fusion protein is insoluble, it can be purified by affinity
chromatography if the protein is solubilized in a solubilizing agent which does not
disrupt binding to glutathione-agarose, such as 1% Triton X-100,1% Tween 20,10
mM dithiothreitol or 0.03% [NADODSO4. OTHER] methods used to solubilize GST-
fusion proteins are described by Frangioni and Neel (Anal. Biochem. 210: 179-87,
1993, herein incorporated by reference). Glutathione fusion proteins can be
attached to an agarose covered substrate, for example a layer of agarose on the
cylindrical substrate, for example by using the techniques disclosed in Lewis et al.,
Protein Expr. [PRUIF.] 13: 120-126,1998, which is incorporated by reference.
Methods and plasmid vectors for producing fusion proteins and intact
native proteins in bacteria are described in Sambrook et al. (Molecular [CLONING:] A
Laboratory Manual, Cold Spring Harbor, New York, 1989, chapter 17, herein
incorporated by reference). Such recombinant fusion proteins may be made in large
amounts, and are easy to purify. Native proteins can be produced in bacteria by
placing a strong, regulated promoter and an efficient ribosome binding site upstream
of the cloned gene. If low levels of protein are produced, additional steps may be
taken to increase protein production; if high levels of protein are produced,
purification is relatively easy. Suitable methods are presented in Sambrook et al.
(Molecular [CLONING. A LABORATORY MANUAL,] Cold Spring Harbor, New York, 1989)
and are well known in the art. Often, proteins expressed at high levels are found in
insoluble inclusion bodies. Methods for extracting proteins from these aggregates
are described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor, New York, Chapter 17,1989).
Vector systems suitable for the expression of actin fusion genes include
the pUR series of vectors (Ruther and [MULLER-HILL,] [EMBO J.] 2: 1791,1983), pEXl-3
(Stanley and Luzio, [EMBO J.] 3: 1429,1984) and [PMR100] (Gray et al., Proc. [NATL.]
[ACAD.] [SCI.] USA 79: 6598,1982). Vectors suitable for the production of intact native
proteins include pKC30 (Shimatake and Rosenberg, Nature 292: 128,1981),
[PKK177-3] (Amann and Brosius, Gene 40: 183,1985) and pET-3 (Studiar and
Moffatt, J. Mol. Biol. 189: 113,1986). Actin fusion proteins may be isolated from
protein gels, for use in the molecular motor. The DNA sequence can also be
transferred to other cloning vehicles, such as other plasmids, bacteriophages,
cosmids, animal viruses and yeast artificial chromosomes [(YACS)] (Burke et al.,
Science 236: 806-812,1987). These vectors may then be introduced into a variety of
hosts including somatic cells, and simple or complex organisms, such as bacteria,
fungi (Timberlake and Marshall, Science 244: 1313-1317,1989), invertebrates,
plants (Gasser and Fraley, Science 244: 1293,1989), and mammals (Pursel et al.,
Science 244: 1281-1288,1989), which cell or organisms are rendered transgenic by
the introduction of the heterologous actin [CDNA.]
For expression in mammalian cells, the actin [CDNA] sequence may be
ligated to heterologous promoters, such as the simian virus SV40 promoter, in the
pSV2 vector (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78: 2072-2076,1981),
and introduced into cells, such as monkey [COS-1] cells (Gluzman, [CELL 23:] 175-82,
[1981),] to achieve transient or long-term expression. The stable integration of the
chimeric gene construct may be maintained in mammalian cells by biochemical
selection, such as neomycin (Southern and [BERG, J. MOL. APPL. GENET.] 1: 327-41,
1982) and mycophoenolic acid (Mulligan and Berg, [PROC. NATL. ACAD. SCI. USA]
78: 2072-2076,1981).
The [CDNA] sequence (or portions derived from it) or a mini gene (a
[CDNA] with an intron and its own promoter) may be introduced into eukaryotic
expression vectors by conventional techniques. These vectors are designed to permit
the transcription of the [CDNA] eukaryotic cells by providing regulatory sequences
that initiate and enhance the transcription of the [CDNA] and ensure its proper splicing
and polyadenylation. Vectors containing the promoter and enhancer regions of the
SV40 or long terminal repeat (LTR) of the Rous Sarcoma virus and polyadenylation
and splicing signal from SV40 are readily available (Mulligan and Berg, Proc. Natl.
[ACAD.] Sci. USA 78: Gorman et al., Proc. Natl. Acad. Sci USA
78: 6777-6781,1982). The level of expression of the cDNA can be manipulated with
this type of vector, either by using promoters that have different activities (for
example, the baculovirus pAC373 can express cDNAs at high levels in S. [FRUGIPERDA]
cells (Summers and Smith, Genetically Altered Viruses and the Environment, Fields
et al. (Eds.) 22: 319-328, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York, [1985)] or by using vectors that contain promoters amenable to
modulation, for example, the glucocorticoid-responsive promoter from the mouse
mammary tumor virus (Lee et al., Nature 294: 228,1982). The expression of the
actin [CDNA] can be monitored in the recipient cells 24 to 72 hours after introduction
(transient expression).
In addition, some vectors contain selectable markers such as the gpt
(Mulligan and Berg, Proc. [NATL.] [ACAD.] Sci. USA 78: 2072-6,1981) or neo (Southern
and Berg, J. [MOL. APPL. GENET.] 1: 327-41,1982) bacterial genes. These selectable
markers permit selection of transfected cells that exhibit stable, long-term expression
of the vectors (and therefore the cDNA). The vectors can be maintained in the cells
as episomal, freely replicating entities by using regulatory elements of viruses such
as papilloma (Sarver et al., Mol. [CELL BIOL. 1. 486, 1981) OR EPSTEIN-BARR (SUGDEN ET]
[AL.,] Mol. Cell Biol. 5: 410,1985). Alternatively, one can also produce cell lines that
have integrated the vector into genomic DNA. Both of these types of cell lines
produce the gene product on a continuous basis. One can also produce cell lines that
have amplified the number of copies of the vector (and therefore of the [CDNA] as
well) to create cell lines that can produce high levels of the gene product (Alt et al.,
[J.] [BIOL. CHEM.] 253: 1357,1978).
The transfer of DNA into eukaryotic, in particular human or other
mammalian cells, is now a conventional technique. The vectors are introduced into
the recipient cells as pure DNA (transfection) by, for example, precipitation with
calcium phosphate (Graham and vander Eb, Virology 52: 466,1973) or strontium
phosphate (Brash et al., [MOL. CELL BIOL.] 7: 2013,1987), electroporation (Neumann et
al., [EMBO J] 1: 841,1982), lipofection (Felgner et al., Proc. Natl. Acad. Sci. U S A.
84: 7413-7417,1987), DEAE dextran (McCuthan et al., [J.] Natl Cancer [INST. 41:] [351,]
1968), microinjection (Mueller et al., [CELL 15:] 579,1978), protoplast fusion
(Schafner, Proc. [NATL.] [ACAD. SCI.] USA 77: 2163-7,1980), or pellet guns (Klein et al.,
Nature 327: 70,1987). Alternatively, the [CDNA] can be introduced by infection with
virus vectors. Systems are developed that use, for example, retroviruses (Bernstein
et al., Gen. Engrg. 7: 235,1985), adenoviruses (Ahmad et al., J. Virol. 57: 267,1986),
or Herpes virus (Spaete et al., [CELL 30:] 295,1982).
Using the above techniques, the expression vectors containing the actin
gene or [CDNA] sequence or fragments or variants or mutants thereof can be
introduced into human cells, mammalian cells from other species or non-mammalian
cells as desired. For example, monkey COS cells (Gluzman, [CELL 23:] 175-82,1981)
that produce high levels of the SV40 T antigen and permit the replication of vectors
containing the SV40 origin of replication may be used. Similarly, Chinese hamster
ovary (CHO), mouse NIH 3T3 fibroblasts or human fibroblasts or [LYMPHOBLASTS] may
be used.
The recombinant cloning vector, according to this invention, then
comprises the selected DNA of the DNA sequences of this invention for expression
in a suitable host. The DNA is operatively linked in the vector to an expression
control sequence in the recombinant DNA molecule so that the actin polypeptide can
be expressed. The expression control sequence may be selected from the group
consisting of sequences that control the expression of genes of prokaryotic or
eukaryotic cells and their viruses and combinations thereof. The expression control
sequence may be specifically selected from the group consisting of the lac system,
the trp system, the tac system, the trc system, major operator and promoter regions
of phage lambda, the control region of fd coat protein, the early and late promoters
of SV40, promoters derived from [POLYOMA.] adenovirus, retrovirus, baculovirus and
simian virus, the promoter for 3-phosphoglycerate kinase, the promoters of yeast
acid phosphatase, the promoter of the yeast alpha-mating factors and combinations
thereof.
EXAMPLE 5
Motor Protein Variants
Variants of the motor proteins (such as actin and myosin) can be used
instead of the native proteins, as long as the variants retain the motor activity. DNA
mutagenesis techniques may be used to produce variant DNA molecules, and will
facilitate the production of proteins which differ in certain structural aspects from
the native protein, yet the variant proteins are clearly derivative and maintain the
essential [FUNCTIONAL] characteristic of the motor protein as defined above. Newly
derived proteins may also be selected in order to obtain variations in the
characteristics of the motor protein, as will be more fully described below. Such
derivatives include those with variations in the amino acid sequence including minor
deletions, additions and substitutions.
While the site for introducing an amino acid sequence variation is
predetermined, the mutation per se need not be predetermined. For example, in
order to optimize the performance of a mutation at a given site, random mutagenesis
may be conducted at a target codon or region and the expressed protein variants
screened for optimal activity. Techniques for making substitution mutations at
predetermined sites in DNA having a known sequence are well known.
Amino acid substitutions are typically of single residues. for example 1,
2,3,4 or more substitutions; insertions usually will be on the order of about from 1
to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
Substitutions, deletions, insertions or any combination thereof may be combined to
arrive at a final construct. Obviously, the mutations that are made in the DNA
encoding the protein must not place the sequence out of reading frame, and
preferably will not create complementary regions that could produce secondary
changes in the [MRNA] structure.
Substitutional variants are those in which at least one residue in the
amino acid sequence has been removed and a different residue inserted in its place.
Such substitutions are generally conservative substitutions when it is desired to
finely modulate the characteristics of the protein. Examples of such conservative
substitutions are well known, and are shown, for example, in U. S. Patent No.
5,928,896 and U. S. Patent No. 5,917,019.
Substantial changes in function or immunological identity are made by
selecting substitutions that are less conservative i. e., selecting residues that differ
more significantly in their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. The substitutions which in general are expected to
produce the greatest changes in protein properties will be those in which (a) a
hydrophilic residue, e. g., seryl or threonyl, is substituted for (or by) a hydrophobic
residue, e. g., leucyl, [ISOLEUCYL,] phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having an electropositive
side chain, e. g., [LYSYL,] arginyl, or histadyl, is substituted for (or by) an
electronegative residue, e. g., glutamyl or aspartyl ; or (d) a residue having a bulky
side chain, e. g., phenylalanine, is substituted for (or by) one not having a side chain,
e. g., glycine.
In view of the many possible embodiments to which the principles of our
invention may be applied, it should be recognized that the illustrated embodiment is
only a particular example of the invention and should not be taken as a limitation on
the scope of the invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that comes within the
scope and spirit of these claims.
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