WO2004011649A1

WO2004011649A1 - Recombined myosin

Info

Publication number: WO2004011649A1
Application number: PCT/JP2003/009741
Authority: WO
Inventors: Kazuhiro Kohama; Akio Nakamura; Hozumi Kawamichi; Hideyuki Tanaka
Original assignee: Kazuhiro Kohama; Akio Nakamura; Hozumi Kawamichi; Hideyuki Tanaka
Priority date: 2002-07-31
Filing date: 2003-07-31
Publication date: 2004-02-05
Also published as: JP4367825B2; JP2004057152A; AU2003254788A1

Abstract

A Ca2+-binding recombinant myosin which is expressed by polynucleotides respectively encoding the heavy chain, the Ca2+-binding light chain and the phosphorylated light chain or Ca2+-biding myosin and has substantially the same function as Ca2+-binding myosin of the wild type.

Description

Description Recombinant Myosin Technical Field

The invention of this application relates to recombinant Ca ²⁺ -linked myosin. More specifically, the present application relates to a Ca ²⁺ -binding recombinant myosin that is expected to be useful as an activator in micromachines and a switch element in microelectronic circuits. Background art

With the recent development of nanotechnology, attention has been focused on the development of micromachines that move mechanically at the molecular size. The creation of this micromachine requires a variety of technological developments, ranging from individual component devices and their assembly methods (micromachining). In particular, the development of the micro-actuator all-in-one, which is a micro-machine driving unit, is indispensable for the autonomous movement of machines, etc., and the development of motor devices using various micromachining technologies is being promoted. However, the size of a microactuator that can be created by applying microfabrication technology is only about Ι ΟΟ でも even if it is small, and further miniaturization is required in order to equip a nanoscale micromachine.

Therefore, it has been proposed to use a single molecule with motor ability as a motor, instead of building a motor-and-motor device using microfabrication technology.

In general, molecules that can be used as motors are required to satisfy two points: that they have a power mechanism that converts external energy into motion, and that they can achieve unidirectional motion. And, as the low molecular weight organic compound satisfying such conditions, (3R, 3 'R)-(P, P) -trans-l, 1', 2, 2 ', 3, 3', 4, 4'-octahydro-3, 3'-dimethyl-4, 4'-bipheanthrydiene (Nature 401: 15 2-155, 1999) and Triptycyl (4) hel icene (Nature 401: 150-152, 1999) are known I have. However, these organic compounds have fatal defects such as extremely low speed, extremely high driving force, and the inability to rotate repeatedly, as an activator in micromachines. There is no prospect of practical application at present. On the other hand, as a single-molecule motor other than the organic compound as described above, a flagellar motor (Microbiol. 6: 1-18, 1967; Nature 245: 380-382, 1973), ATP synthase (Nature 386: 299-302, 1997), myosin motor (Biochem. Biophys. Res. Comm. 199: 1057-1063, 1994; Curr. Opin. Cell Biol. 7: 89-93, 1995), microtubule motor ( Cell 42: 39-50, 1985) and biomolecules such as motor proteins of nucleic acid synthase (Nature 409: 113-119, 2001) are known.

Of course, these biomolecules require various technological developments in order to be used for micromachine activites, but due to their stable driving, etc., they can become extremely powerful devices in future micromachines. Is expected. In addition, it has been proposed that these biomolecules can also be used as switch elements in electronic circuits. Of these biomolecule monitor candidates, myosin is a muscle protein with a molecular weight of about 480 kDa, two myosin heavy chains with a molecular weight of about 220 kDa, and two myosin light chains, two each. Consists of chains. The N-terminal side of the myosin heavy chain is a functional site called heavy meromyosin (HMM), which functions as a protein that generates “force” between actin and ATP by degradation of ATP.

There are two types of myosin regulation: (a) types that are not externally regulated, such as skeletal muscle myosin, (b) types that are regulated by phosphorylation, such as smooth muscle and cellular slime mold Dictyostelium myosin, and (c) ) Calcium (Ca ²⁺ ), like the myosin of the slime mold Physarum (sea lop) There are types that are controlled by combining

As mentioned above, the usefulness of biomolecules as micromachine actuaries (molecular motors) and switch elements in electronic circuits has been pointed out. And each biomolecule can be applied and combined in various ways depending on its individual properties (difference in rotation, driving mechanism, etc.).

On the other hand, in order to use these biomolecules as mechanical components such as actuator devices, various modifications (such as various types) that enable mechanical and electrical coupling with other devices are required. Modifier) is required. The most effective means for ensuring such modification is to genetically modify the biomolecule.

In this regard, in the case of myosin, the production of recombinant myosin has been reported for the types (a) and (b) (for example, Pro Natl. Acad. Sc. USA 92: 704-708). , 1995; Science 246: 656-658, 1989). However, recombinant production of Ca ²⁺ -linked myosin of type (c) has not been successful.

The invention of this application has been made in view of the above circumstances, and has an object to provide a Ca ²⁺ -binding recombinant myosin. Disclosure of the invention

This application, as the invention for solving the above problems, there the heavy chain, Ca ²⁺ binding light chain, and the expression product of a polynucleotide encoding the respective phosphorylation light chain of Ca ²⁺ bound Mi Oshin Thus, there is provided a recombinant myosin characterized by having substantially the same function as a wild-type Ca ²⁺ -binding myosin.

In another embodiment of the present invention, one or more amino acid residues in the amino acid sequence of the myosin heavy chain, Ca ²⁺ binding light chain and / or phosphorylated light chain are substituted with other amino acid residues. Is a recombinant myosin in which is deleted or one or more amino acid residues are added. In a preferred embodiment of the recombinant myosin of the present invention, each polynucleotide is derived from a true slime mold Physalum. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of SDS-PAGE of the recombinant S1-heavy chain expressed in Si-9 cells. Lane 1 is the molecular weight marker, Lane 2 is the homogenate of uninfected Sf-9 cells, Lane 3 is the precipitate of infected Sf-9 cell homogenate, and Lane 4 is the supernatant of infected Si-9 cell homogenate.

FIG. 2 shows the results of SDS-PAG in the S1 purification process. Lane 1 is the separation marker, Lane 2 is the homogenized and centrifuged supernatant of uninfected Si-9 cells, Lane 3 is the sediment of the same cells, Lane 4 is S1-heavy chain, phosphorylated light chain and Ca Supernatants obtained by homogenizing and centrifuging Si-9 cells infected with the 2 ^{+ -linked} light chain, Lane 5 is the precipitate of the same cells, Lane 6 is S1 purified from the complex of S1 and actin, Lane 7 is S 1 purified by a Ni-NTN column.

FIG. 3 shows the results of SDS-PAGE during the recombinant MM purification process. Lane 1 is the molecular weight marker, Lane 2 is the centrifuged supernatant of uninfected Sf-9 cell homogenate, Lane 3 is the precipitate, and Lane 4 is the S heavy, phosphorylated and Ca2 ⁺ light chains. The supernatant of the infected Si-9 cell homogenate, Lane 5 is the precipitate of the infected cells, Lane 6 is the sample before the Ni-NTA column, and Lane 7 is the purified HMM eluted from the Ni-NTN column.

FIG. 4 is an electron micrograph of the recombinant HMM purified in the step of FIG. FIG. 5 shows the measurement results of ATPase activity of S1. 1 is 0. Reference Mg ²⁺ -ATPase activity in the presence of ImM EGTA, 2 is 0. Actin-activating ATPase activity in the presence of ImM EGTA and 5 M actin, 3 is 0. 0 in the presence of ImM Ca ²⁺ Actin activation ATPase activity. Values are the average soil SEM of three measurements. FIG. 6 shows the results of measuring ATPase activity of S1 in the presence of various concentrations of actin. The concentrations of S1 and EGTA were 0. and 0. ImM, respectively. Specified. Other measurement conditions were the same as in FIG.

FIG. 7 shows the results of measuring the ATPase activity of the recombinant HMM. 1 is 0. Reference Mg ²⁺ -ATPase activity in the presence of ImM EGTA, 2 is 0. Actin-activating ATPase activity in the presence of ImM EGTA and actin, 3 is 0. 0 in the presence of ImM Ca ²⁺ and actin Actin-activated ATPase activity. Values are the mean SEM of three measurements.

FIG. 8 shows the results of measuring the ATPase activity of recombinant HMM in the presence of various concentrations of actin.

FIG. 9 shows the results of measuring the calcium binding activity of the recombinant HMM. The closed circle is HMM, the open circle is CaLC, and the closed diamond is PLC.

FIG. 10 shows the results of measuring the effect of Ca ^{2+ on} the motor activity of HMM. A is the measurement result in the presence of 0. ImM EGTA, and B is the measurement result in the presence of 0. ImM Ca2 +. Arrows indicate the average rate of ATP-dependent movement of actin. BEST MODE FOR CARRYING OUT THE INVENTION

Recombinant myosin of the invention, the heavy chain of the Ca ²⁺ binding myosin (about 220 kDa), Ca ²⁺ binding light chain (approximately 16 kDa), and the polynucleotides encoding each of the phosphorylated light chains (about 18 kDa) It is an expression product.

― “Ca ²⁺ binding myosin” is a myosin that is regulated by binding Ca ^{i +} , specifically, myosin of Physarum (Physalum) or Scallop (scal lop). is there. Further, "has substantially the same function as wild-type Ca ²⁺ -linked myosin" means that myosin degrades ATP and binds to actin to generate mechanical energy. This means that Ca ²⁺ gives a change. However, recombinant myosin from Physalum reduces ATP degradation and generation of mechanical energy by Ca2 ⁺ binding, and recombinant myosin from scallop reduces ATP degradation and generation of mechanical energy by Ca2 ⁺ binding. Means to increase.

The recombinant myosin of the present invention comprises: It can be prepared by expression in a translation system or an appropriate host vector system.

For example, the polynucleotide (cDNA) can be obtained from publicly known base sequence information (eg, myosin heavy chain of Physalum: GenBank No. AF335500 Ca ²⁺ binding light chain: GenBank No. 03499, phosphorylated light chain: GenBank No. AB076705; scallop) Myosin heavy chain: GenBank No. X55714, light chain: GenBank No. M17208, M17201) can be obtained by screening each cDNA library using an oligonucleotide synthesized based on the same. Also, the desired polynucleotide can be obtained by PCR or RT-PCR using an oligonucleotide primer prepared based on known nucleotide sequence information.

The obtained polynucleotides are separately cloned into expression vectors and co-expressed, whereby the recombinant myosin of the present invention can be prepared. Alternatively, each polynucleotide may be cloned into an expression vector as a fusion polynucleotide in which the respective polynucleotides are linked. However, in this case, a stop codon should be provided at the 3 'end of each polynucleotide so that the heavy chain, Ca2 + -binding light chain and phosphorylated light chain are each expressed as a mature protein. Also, the 5 'end of each polynucleotide may be provided with a respective expression control sequence (promoter-no enhancer).

When the recombinant myosin of the present invention is expressed in an in vitro translation system, the polynucleotide is recombined into an expression vector having an RNA polymerase promoter, and this recombinant vector is used for the promoter. It is added to an in vitro translation system, such as a reticulocyte lysate of egrets containing RNA polymerase or a wheat germ extract. Examples of the RNA polymerase promoter include T7 and T3 SP6. Examples of vectors containing these RNA polymerase promoters include pKAK pCDM8, ΡΤ3 / Τ718, ρΤ7 / 319, pBluescript II and the like. In addition, if the polynucleotide is expressed in an appropriate host vector system, the recombinant myosin can be used for prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, mammalian cells, and plant cells. Etc. can be produced. For example, when expressing in a microorganism such as Escherichia coli, a polynucleotide is assembled in an expression vector having an origin, a promoter, a ribosome binding site, a DNA cloning site, and a chromosome that can replicate in the microorganism. If an expression vector is prepared by reconstitution, a host cell is transformed with the expression vector, and this transformant is cultured, the target recombinant myosin can be produced in large quantities from the culture. Examples of expression vectors for Escherichia coli include a plIC system, pBluescript II, a pET expression system, a pGEX expression system, and the like. Furthermore, when recombinant myosin is expressed in eukaryotic cells, the polynucleotide is inserted into an expression vector for eukaryotic cells having a promoter, a splicing region, and a poly (A) -added site. A desired recombinant myosin can be obtained from a eukaryotic cell prepared by preparing a vector and transfecting the vector. Examples of expression vectors include pKAl, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2 and the like. As eukaryotic cells, mammalian cultured cells such as human embryonic kidney cells HEK293, monkey kidney cells C0S7, Chinese hamster ovary cells CH0, or primary cultured cells isolated from human organs can be used. Saccharomyces cerevisiae, fission yeast, silkworm cells, African algae eggs and the like can also be used. Further, if the expression vector is transfected into insect cells together with the genomic DNA of nuclear polyhedrosis virus belonging to the baculovirus family, the desired recombinant myosin can be obtained from the insect cells. Si 9, S 1, TD5 and the like can be used as insect cells.

To introduce the expression vector into cells, known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used. In order to isolate and purify the polypeptide expressed in the transformed cell, known separation operations can be combined. For example, urea Treatment with any denaturant or surfactant, sonication, enzymatic digestion, salting out / solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, ion exchange Chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, and the like.

Another embodiment of the present invention provides a method for preparing a myosin heavy chain, a Ca2 ⁺ binding light chain and / or a phosphorylated light chain, wherein one or more amino acid residues in the amino acid sequence is replaced with another amino acid residue, Modified recombinant myosin in which residues are deleted or one or more amino acid residues are added.

In this case, the “modified type” means that the activity of the recombinant myosin (eg, Ca ²⁺ binding ability, ATP resolution, mechanical energy generation ability, etc.) is increased or decreased due to the amino acid sequence mutation. Means Alternatively, it refers to an amino acid mutation that increases or decreases the binding to other molecules or compounds.

Such an amino acid mutation can be performed by introducing a mutation into the above-described polynucleotide by a known method, and expressing the mutant polynucleotide in the same manner as described above. For example, when preparing a polynucleotide in which an arbitrary amino acid codon is replaced by another amino acid codon, the known Kunkel method (Kunkel, TA Pro Natl. Acad. Sci. USA 82: 488, 1985 and Kunkel, TA, et al. Methods in Enzymology 154: 367, 1987). That is, Escherichia coli (BW313, CJ236, etc.) represented by the dut- and ung- genotypes are deficient in dUTPase (Dut) and Uraci DNA glycosylase (Ung). It synthesizes DNA in which part of T) has been replaced by deoxyduracil (dU). Using this Escherichia coli as a host bacterium, an oligonucleotide designed to replace the target residue with another amino acid residue is hybridized in a test tube to a target polynucleotide for mutation, and the DNA polymerase reaction is performed. A complementary DNA strand is synthesized by a DNA ligase reaction. This DNA is ung + E. coli strain When introduced into (DH5a, etc.), the original DNA strand containing dU undergoes degradation by Uiig, but the complementary DNA strand synthesized in the test tube is replicated without degradation. In this manner, the DNA strand on which the mutation has been introduced is selectively amplified, and a polynucleotide encoding myosin containing the target mutation can be obtained. Mutant polynucleotides can be obtained by a method using mutation kit or the like, a mutation-introduced PCR method, or a known polynucleotide synthesis method (for example, Nucleic Acid Res. 25: 3440-3444, 1997 etc.).

Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited to the following examples. Example

(1) Materials and methods

(1-1) Chemical substances

Restriction enzymes and other enzymes were from Takara Shuzo Co., Ltd. (Kyoto). All other reagents were commercially available special grade reagents. Milli-Q water (Millipore, Bedford, MA, USA) was used when preparing the aqueous solution.

(1-2) Construction of baculovirus introduction vector

CDNA encoding the subfragment-1 heavy chain (S I-HC; Metl-Gly841) of myosin heavy chain derived from physalum was synthesized by RT-PCR (KOD DNA polyierase; Toyobo Co., Ltd., Tokyo). PCR primers have known sequence information (GenBank No. AF335500)

The following, which was designed based on, was used.

5'-CGGGATCCATGGCAAGCGAAAGGCAAC-3 '(SEQ ID NO: 1)

5′-ATGGTGCTTGTCGTCGTCGTCGCCAACCAATAAGGGACGCG-3 ′ (SEQ ID NO: 2) As the PCR conditions, 35 cycles were performed, with one cycle consisting of denaturation (1 minute at 94), annealing (55 ti minutes), and extension (723 minutes). In order to express recombinant myosin, the sequence of the hexa-His-tag was added to the C-terminus by PCR. The following primers at the end of S1-HC: 5'-CCGCGGCCGCATGATGATGATGATGGTGCTTGTCGTCGTCGTCGTC-3 '(SEQ ID NO: 3) contained a hex His tag sequence, a stop codon and a SaiI restriction site.

Nest PCR was performed using the following primers.

5'-CGGGATCCATGGCAAGCGAAAGGCAAC-3 '(SEQ ID NO: 4)

The PCR product obtained above was cloned into the BamHI / Sall site of pBlueBac4.5a (Invitrogen, Carlsbad, Calif., USA) to confirm the DNA sequence. The obtained plasmid was designated as pBB / Sl.

The vector for the introduction of the heavy chain of myomesin meromyosin (HMM) heavy chain (MeU-Lysll81) was constructed in the same manner as described above. A coiled-coil structure region, which is a part of the myosin heavy chain, was added to the C-terminus of the HC by PCR.

Primer with Smal restriction site (2044 bp of myosin heavy chain):

5'-CAGAAGCCCGGGTACCTTG-3 '(SEQ ID NO: 5) and

Primers containing an HMM fragment (Lysll81) at the end:

5'-ATGATGATGGTGCTTGAGCTCCTCTACCTG-3 '(SEQ ID NO: 6)

Was used. To add a HexaHis tag sequence, primer:

5'-CAGAAGCCCGGGTACCTTG-3 '(SEQ ID NO: 7) and

Primers containing the HexaHis tag sequence, stop codon, and Sail restriction site:

Nest PCR was performed using 5′-GGACTAGTGTCGACTTAATGATGATGATGATGGTGC-3 ′ (SEQ ID NO: 8).

The obtained PCR product was cloned into Smal / Sall of pBB / S1 plasmid, and the plasmid was constructed into pBB / HMM.

Furthermore, using primers designed based on the known sequence information, vectors for introducing polynucleotides encoding the Ca ²⁺ binding light chain (CaLC) and the phosphorylated light chain (PLC) were constructed. In addition, BamHI or Kpnl restriction enzyme sites were added to the 5 'end of PLC and CaLC. Also PLC and A Kpnl or EcoRI site was added to the 3 'end of CaLC. The resulting PCR products were subcloned into BamHI / Kpnl and Kpnl / EcoRI restriction sites of pBlueBac4.5a, respectively, to construct plasmids pBB / PLC and pBB / CaLC.

-3) Infection and selection of recombinant virus

Spodoptera frugiperda (Sf-9) cells were 27 in 75 cm2 flasks.

The culture solution (Tand-FH) was prepared by adding 10% fetal calf serum (FCS) and 10 g / ml gentamicin (Sigma-Aldricli, USA) to Grace's insect cell culture solution (Invitrogen, Carlsbad, CA, USA). Was added. In Si-9 cells, the linear DNA (Bac-M-BlueTM DNA; Invitrogen, Carlsbad, CA, USA) of the nuclear polyhedrosis virus Auto-graphica californica, the transfer vectors pBB / Sl, pBB / HMM, pBB / Infections were introduced with either PLC or pBB / CaLC. InsectinPlus ™ ribosome reagent (Invitrogen, Carlsbad, CA, USA) was used to increase the efficiency of infection transfer. Seven days later, a black assay was performed on an X-gal-containing plate to isolate a recombinant paculovirus. We picked up the blue break and infected Si-9 cells (25 cm ² culture vessel) to amplify the recombinant paculovirus. Four days later, the recombinant Paculovirus in the supernatant was used to infect Si-9 cells cultured in the above-mentioned 75 cm ² flask for 11 days to prepare a stock having a high virus titer. Various introduced cDNAs integrated into the recombinant PacuMouth virus were confirmed by PCR, and subsequently also confirmed by a nucleotide sequence analyzer. The obtained recombinant paculovirus was propagated again by the method described above.

(4) Purification of recombinant S1 and HMM

1 × 10 ⁷ Si-9 cells were inoculated in each of 10 culture vessels of 72 cm ² . Si-9 cells were individually co-infected with Pacu Mouth virus into which polynucleotides encoding S heavy chain or HMM-heavy chain, phosphorylated light chain and Ca ²⁺ binding light chain, respectively, were introduced. In each case, the severity of infection (m.0.i) was 5. Infected Si-9 cells were grown at 27 for 3 days. After elongation, the cells were centrifuged at 4 rpm for 10 minutes at 1500 rpm to collect the cells. Since the collected cells are in pellet form, 7 ml of homogenizing buffer (20 mM Tris-HCl (pH 7.5), ImM MgCl ₂ , ImM EGTA, 5iM 2 mercaptoethanol, protease inhibitor lmMp-ABSF (Wako Pure Chemical Industries, Ltd.) Industrial Co., Ltd.), lOg / ml mixture of leptin and His-tagged protease inhibitor (Sigma-Aldrich, USA)]. After homogenization, the recombinant myosin was released from endogenous actin, adjusted to 0.2 M NaCl and ImM ATP, and centrifuged at 15000Xg for 15 minutes to remove cell debris and unbroken cells. . The supernatant was mixed with 0.2 mg / ml ゥ heron bone skeletal muscle actin (purified from ゥ heron skeletal muscle acetone powder) and buffered (20 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 iMMgC12, 0.3 mM DTT, ImM p-ABSF and (1 ig / ml of leptin) for 24 hours. Actin recombinant HMM was formed and precipitated during dialysis, and collected by centrifugation with lOOOOOXg for 1 hour. The precipitate separated S1 / HMM than precipitate buffer [20mM Tris-HCl (pH 7.5 ), lOOmM KCK lOmM MgCl 2, 7mM 2- mercaptoethanol, IOO MP - ABSF, 1 g / ml Roy [Peptin and ImM ATP]], centrifuged with lOOOOOXg for 90 minutes, and the resulting supernatant was applied to a Ni-NTA spa flow (Qiagen, Germany) column. In this column, trap His-tagged recombinant S1 'or HMM, extract i20mM Tris-HCl (pH 7.5), 40mM KCK 7mM 2-mercaptoethanol, I'g / ml leptin and 100mM imidazole ]. After SDS-PAGE, the electrophoretic band containing the recombinant S1 / HMM was accumulated and concentrated using a Centriprep-30 concentrator (Millipore, USA). The recombinant myosin was then permeated with buffer UOmMTris-HC1 (pH 7.5), 40 mM KCK 1.5 mM MgCl ₂ , 0.1 lmM DTT, 20 M p-ABSF and 0.6 g / ml leptin. The protein was purified under the following temperature conditions. As a result of collecting 0.1 mg of typical recombinant S1 / HMM and quantifying it with Bio-Rad protein measurement solution using BSA as a standard value, 1 × 10 ⁸ Si-9 cells were obtained. (1-5) gel electrophoresis

SDS-PAGE was performed using a 12.5¾ polyacrylamide gel prepared using the buffer system described in the literature (Nature 227: 680-685, 1970) according to the description in the literature (J. Chromatogr 64: 147-155, 1972). It was carried out using. The SDS-sample buffer was composed of 40 mM Tris-HCl (pH 6.8), 50 mM DTT, 1¾ SDS, 7.5 glycerol, and 0.002 bromophenol blue.

(1-6) Observation by electron microscope

The purified HMM (0.5 mg / ml) was placed on a mica sheet, washed three times with 0.1 M ammonium acetate containing 30% glycerol, stained with aqueous peranyl acetate, and washed three times. The mica sheet was covered with another divided mica sheet, pressed and peeled off, and then duplicated one by one on platinum at a low angle in a BAF 060 rotary image system. Observed with a JEM-1010 electron microscope.

(1-7) Analysis by ATPase activity

The ATPase activity in S1 / HMM was measured according to the description in the literature (Anal Biochem. 293: 212-215, 2001). All analyzes were performed at 25. Basic Mg ²⁺ -ATPase activity is 20 mM Tris-HCl (pH 7.5), 40 mM KCK 1.5 mM MgCl ₂ , 0.1 liM DTT, 0.5 mM ATP, 重合 skeletal muscle derived actin and 0.5 M recombinant Analysis was performed with S1 / HMM. Statistical analysis was performed using Student's t-test, and P-0.05 was considered statistically significant.

(l-8) Measurement of Ca ²⁺ binding

As described in the literature (Biochemistry 39: 3827-3834, 2000), the binding range of Ca ^{2+ was determined} for 3.5 M HMM, 50 M recombinant Ca ²⁺ light chain or 50 ^ M recombinant phosphorylated light chain. In the presence, it was measured at 25 using a flow dialysis method using 0.1 mM LiCl NaCK containing 5 mM MgCl ₂ containing MoCl ₂ (Du-Pont-ΝΕΝ) and MOPS / NaOH (pH 7.0). The Ndel restriction enzyme site was added to the 5 'end of the 0RF of each phosphorylated light chain or Ca ²⁺ light chain, and the BamHI site was added to the 3' end by PCR. This PCR product is treated with Ndel and BamHI, and the pET19b expression vector One system (Novagen, USA) was purchased at the same site. PETl¾ / PLC or pET19b / CaLC was expressed and purified in E. coli BL21 (DE3) according to the manufacturer's instructions. All data were analyzed by fitting to the Adiar equation.

(1-9) In Vitro Analysis of Motor Force

The analysis of exercise force in vitro was performed as described in the literature (J. Biochem. 106: 955-957, 1989). That is, the recombinant HMM was coated on the glass surface. Polymerized actin (0.125 mg / ml) was labeled with rhodamine phalloidin (Molecular Probe, USA), and the medium for kinetic analysis [10 mM KCl, 2 mM ATP, liM MgC12, lOmM imidazole (pH 7.5), 14 mM 2-mercap Triethanol and 0.1 lmM EGTA or 0.1 lmM CaCl ₂ ]. ATP-dependent movement was observed with a fluorescence microscope and recorded with a video camera. The speed of actin movement was calculated from the distance traveled and the time elapsed when the movement was captured with a video camera. Statistical analysis was performed using Student's t-test, and P-0.05 was considered statistically significant.

(2) Results

Si-9 cells were infected with recombinant S1-heavy chain baculovirus. After 3 days of culture, these cells were collected, homogenized, and centrifuged. The expression product was in the precipitate (FIG. 1), indicating that the S1-heavy chain was insoluble. Similarly, the expression product was obtained as an insoluble substance upon co-infection introduction of the S1-heavy chain baculovirus and the Ca ²⁺ binding light chain paculovirus. However, this co-infection with the recombinant S1-heavy chain baculovirus, the phosphorylated light chain recombinant paculovirus and the Ca2 ⁺ -linked light chain recombinant baculovirus was not effective in Sf-9 cells. Soluble S heavy chains were produced (Fig.

2). Similarly, in the case of HMM-heavy chain, co-infection of the phosphorylated light chain and Ca ²⁺ -linked light chain with the recombinant baculovirus resulted in a soluble product (Fig.

3).

Recombinant Pacu mouth will for S heavy chain, phosphorylated light chain and Ca ²⁺ binding light chain To recover the soluble S1-heavy chain from the raw extract of infected Si-9 cells, large amounts of actin were mixed with this extract and centrifuged. The resulting pellet was suspended in a buffer containing ATP, and the heavy chain was released from the pellet. The remaining actin was removed by centrifugation again, and the obtained supernatant was injected into a Ni-NTA spa flow column. As shown in FIG. 2, the extract is composed of three large bands, 98 kDa (S1-heavy chain), 18 kDa (phosphorylated light chain), and 16 kDa (Ca ²⁺ binding light chain).

This indicates that the extract contains S1. HMM is similar to the complex of HMM-heavy chain peptide 135 kDa, phosphorylated light chain peptide 18 kDa and Ca ²⁺ binding light chain peptide 16 kDa (FIG. 3). A double-headed HMM was shown by electron microscopy (Figure 4). The ATPase activity of S1 and HMM was analyzed by high performance liquid chromatography, which allows quantification of even small samples. ATPase activity was confirmed in recombinant S1, and this activity was activated by actin as follows. That is, Mg ²⁺ -ATPase activity of 0.12 soil 0.01 (s-lhead-1) (n = 3) (m soil SEM) (Fig. 5);

In the presence of various concentrations of actin, the activity increased to Vmax = 0.61 (s-lhead-1) together with Km = 2.5 M (Fig. 6). Thus, it was confirmed that S1 was activated up to about 5-fold by actin, and that functional S1 was expressed. HMM also showed Mg ²⁺ -ATPase activity of 0.21 ± 0.02 (s-lhead-1) (n = 3) (m soil SEM) (FIG. 7). Actin increased the activity to Vmax = 1.27 (s-lhead-1) by Km = 1.8 M (Fig. 8), confirming that HMM was activated by actin to about 6-fold. .

The effect of Ca ^{2+ on} S1 ATPase activity was tested under maximal activation by actin. In the presence of EGTA, a chelator of Ca ²⁺ , the activity was 0.49 ± 0.07 (s-lhead-1) (n = 3). 0. In the presence of ImM Ca ²⁺ , the activity decreased slightly to 0.45 ± 0.04 (s-lhead-1) (n = 3) (Fig. 5). However, this decrease was not statistically significant. The effect of Ca ²⁺ was similarly tested on the HMM. But the effect, if any, is It was a quiet thing (Figure 7).

Since the effect of Ca ²⁺ is not detected, the question occurs as to whether S1 and HMM is bound to Ca ^2+. Therefore, Ca ²⁺ binding by HMM was confirmed by the flow dialysis method. As shown in FIG. 9, the phosphorylated light chain did not bind to Ca ²⁺ . However, Ca ²⁺ binding light chain was confirmed to have Ca ²⁺ binding activity.

HMM binding activity increased dramatically;

The maximum binding activity was 2 mol / iDol HMM with Kd at the IOM level. The effect of Ca ²⁺ binding on recombinant HMM was examined using in vitro kinetic analysis. That is, ATP-dependent movement of actin was observed on the HMM-coated glass surface (Fig. 10). The average velocity in the presence of EGTA was 0.61 ± 0.16 m / sec (n = 25), while it decreased to 0.32 ± 0.21 m / sec (n = 25) in the presence of Ca ²⁺ . From these results, it was confirmed that the recombinant HMM had Ca ²⁺ binding activity, and that Ca ²⁺ had an effect on the motor function activity of the recombinant HMM. Industrial applicability

As described in detail above, according to the invention of this application, a Ca ²⁺ -linked

Is provided. This recombinant myosin is useful as an activator in a micromachine or as a switch element in an electronic circuit.

Claims

The scope of the claims

1. The heavy chain of Ca ²⁺ binding myosin, an expression product of a polynucleotide encoding Ca ²⁺ binding light chain, and each of phosphorylated light chain, substantially the Ca ²⁺ binding myosin wild type Recombinant myosi characterized by having the same function

N.

2. One or more amino acid residues in the amino acid sequence of myosin heavy chain, Ca ²⁺ binding light chain and / or phosphorylated light chain are replaced with other amino acid residues, one or more amino acid residues are deleted, Or the recombinant myosin according to claim 1, wherein one or more amino acid residues are added.

3. The recombinant myosin according to claim 1 or 2, wherein each polynucleotide is derived from Escherichia coli Physalum.

Seauence Listing ku 110> Kohama, Kazuhiro,

Nakamura, Akio

Kawamichi, Hozumi

Tanaka, Hideyuki

<120> Recombinant Myosin

<130> NP02332-YS

<160>

<210> 1

C 211> 27

<212> DNA

<213> Artificial sequence

<220>

223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 1

CGGGATCCAT GGCAAGCGAA AGGCAAC 27 ku 210〉 2

<211> 41

212> DNA

K 213〉 Artificial sequence

K 220>

223> Description of Artificial Sequence: Synthetic oligonucleotide

ATGGTGCTTG TCGTCGTCGT CGCCAACCAA TAAGGGACGC G 41 <210> 3

C 211> 46

K 212> DNA

<213> Artificial sequence

K 220>

223> Description of Artificial Sequence: Synthetic oligonucleotide 00> 3

CCGCGGCCGC ATGATGATGA TGATGGTGCT TGTCGTCGTC GTCGTC 46 <210> 4

K 211> 27

<212> DNA

K 213〉 Artificial sequence

<220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

CGGGATCCAT GGCAAGCGAA AGGCAAC 27 <10> 5

C 211> 19

<212> DNA

Gu 213> Artificial sequence

<220>

223> Description of Artificial Sequence: Synthetic oligonucleotide <Shelf> 5

CAGAAGCCCG GGTACCTTG 19 ku 210〉 6

K 211〉 30- K 212〉 DNA

<213> Artificial sequence

K 220>

<223> Description of Artificial Sequence: Synthetic oligonucleotide

ATGATGATGG TGCTTGAGCT CCTCTACCTG 30 ku 210〉 7

C 211> 19

K 212> DNA

K 213〉 Artificial sequence

<220>

Ku 22-3> Description of Artificial Sequence: Synthetic oligonucleotide ku 400> 7

CAGAAGCCCG GGTACCTTG 19 <210> 8

C 211> 36

K 212> DNA

213> Artificial sequence

K 220>

223> Description of Artificial SeQuence: Synthetic oligonucleotide <400> 8

GGACTAGTGT CGACTTAATG ATGATGATGA TGGTGC 36