JP4367825B2 - Recombinant myosin - Google Patents

Description

【図面の簡単な説明】
【図１】 Sf-9細胞で発現させた組換えS1-重鎖のSDS-PAGEの結果である。レーン１は分子量マーカー、レーン２は非感染Sf-9細胞のホモジネート、レーン３は感染Sf-9細胞ホモジネートの沈殿物、レーン４は感染Sf-9細胞ホモジネートの上清である。
【図２】 S1精製過程でのSDS-PAGEの結果である。レーン１は分離量マーカー、レーン２はホモジナイズし、遠心分離した非感染Sf-9細胞の上清、レーン３は同細胞の沈殿物、レーン４はS1-重鎖、リン酸化軽鎖およびCa²⁺結合軽鎖を感染させたSf-9細胞をホモジナイズし、遠心分離した上清、レーン５は同細胞の沈殿物、レーン６はS1とアクチンとの複合体から精製したS1、レーン７はNi-NTNカラムにより精製したS1である。
【図３】組換えHMM精製過程でのSDS-PAGEの結果である。レーン１は分子量マーカー、レーン２は非感染Sf-9細胞ホモジネートの遠心分離上清、レーン３はその沈殿、レーン４はS1-重鎖、リン酸化軽鎖およびCa²⁺結合軽鎖を感染させたSf-9細胞ホモジネートの上清、レーン５は同感染細胞の沈殿、レーン６はNi-NTAカラム前のサンプル、レーン７はNi-NTNカラムから溶出した精製HMMである。
【図４】図３の工程で精製した組換えHMMの電子顕微鏡写真である。
【図５】 S1のATPase活性の測定結果である。１は0.1mM EGTA存在下での基準Mg²⁺-ATPase活性、２は0.1mM EGTAおよび5μMアクチン存在下でのアクチン活性化ATPase活性、３は0.1mM Ca²⁺存在下でのアクチン活性化ATPase活性である。値は３回の測定の平均±SEMである。
【図６】様々な濃度のアクチン存在下におけるS1のATPase活性測定結果である。S1およびEGTAの濃度はそれぞれ0.5μMおよび0.1mMに固定した。他の測定条件は図５と同様とした。
【図７】組換えHMMのATPase活性の測定結果である。１は0.1mM EGTA存在下での基準Mg²⁺-ATPase活性、２は0.1mM EGTAおよび5μMアクチン存在下でのアクチン活性化ATPase活性、３は0.1mM Ca²⁺および5μMアクチン存在下でのアクチン活性化ATPase活性である。値は３回の測定の平均±SEMである。
【図８】様々な濃度のアクチン存在下における組換えHMMのATPase活性測定結果である。
【図９】組換えHMMのカルシウム結合活性の測定結果である。黒丸はHMM、白丸はCaLC、黒菱型はPLCである。
【図１０】 HMMの運動活性に対するCa²⁺の効果を測定した結果である。Aは0.1mM EGTA存在下での測定結果、Bは0.1mM Ca²⁺存在下での測定結果である。矢印はアクチンのATP依存性運動の平均速度を示す。[0001]
BACKGROUND OF THE INVENTION
The invention of this application is Ca ²⁺ The present invention relates to a conjugated recombinant myosin. More specifically, this application is expected to be useful as an actuator in a micromachine or a switch element in a microelectronic circuit. ²⁺ The present invention relates to conjugated recombinant myosin.
[0002]
[Prior art]
With the development of nanotechnology in recent years, attention has been focused on the development of micromachines that move mechanically with molecular size. The creation of this micromachine requires various technological developments up to the individual element devices and their assembly methods (micromachining). In particular, the development of a microactuator that is a micromachine driving unit is indispensable for autonomous movement of a machine and the like, and development of a motor device using various fine processing techniques is being promoted. However, microactuators that can be made by a method that applies microfabrication technology are about 100 μm even if they are small, and further miniaturization is required to equip them with nanoscale micromachines.
[0003]
Therefore, it has been proposed to use a single molecule having motility as a motor, rather than constructing a motor device by microfabrication technology.
[0004]
In general, a molecule that can be used as a motor is required to satisfy two points: that there is a power mechanism that converts external energy into motion, and that a motion in one direction can be realized. And, as a low molecular organic compound satisfying such conditions, (3R, 3'R)-(P, P) -trans-1,1 ', 2,2', 3,3 ', 4,4'-octahydro-3,3'-dimethyl-4,4'-bipheanthrydiene (Nature 401: 152-155, 1999) and Triptycyl (4) helicene (Nature 401: 150-152, 1999) are known. However, these organic compounds have fatal defects as actuators in micromachines such as extremely low speed, maximum driving force, and inability to rotate repeatedly. Is not standing.
[0005]
On the other hand, as a single molecule motor different from the above organic compounds, 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) 1985, 1985), and kinetic proteins of nucleic acid synthase (Nature 409: 113-119, 2001) are known.
[0006]
Of course, these biomolecules require various technological developments in order to be used for micromachine actuators, etc., but their stable driving force is expected to make them extremely powerful devices for future micromachines. Has been. In addition, it has been proposed that these biomolecules can also be used as switching elements for electronic circuits.
[0007]
Among these biomolecular motor candidates, myosin is a muscle protein with a molecular weight of about 480 kDa, consisting of two myosin heavy chains with a molecular weight of about 220 kDa, and two myosin light chains, two each. Yes. The N-terminal side of the myosin heavy chain is a functional site called heavy meromyosin (HMM), which functions as a motor protein that generates “force” with actin by the degradation of ATP.
[0008]
There are two types of myosin: (a) a type that is not externally controlled, such as skeletal muscle myosin, (b) a type that is controlled by phosphorylation, such as smooth muscle and the cellular slime mold Dictyostelium myosin, and (c) Calcium (Ca) like myosin of true slime mold Physarum and scallop ²⁺ There are types that are controlled by combining).
[0009]
[Problems to be solved by the invention]
As described above, the usefulness of biomolecules as actuators (molecular motors) for micromachines and switch elements for electronic circuits has been pointed out. Each biomolecule can be applied and combined in various ways depending on individual characteristics (rotation, difference in driving mechanism, etc.).
[0010]
On the other hand, in order to use these biomolecules as mechanical components such as actuators and switch elements, various modifications (various modifications) that enable mechanical and electrical coupling with other devices are required. is necessary. And the most effective means for reliably performing such modification is to modify biomolecules by genetic engineering.
[0011]
In this regard, in the case of myosin, the production of recombinant myosin has been reported for types (a) and (b) above (eg Proc. Natl. Acad. Sci. USA 92: 704-708, 1995; Science 246: 656-658, 1989). However, type (c) Ca ²⁺ For conjugated myosin, recombinant production has not been successful.
[0012]
The invention of this application has been made in view of the circumstances as described above. ²⁺ It is an object to provide a conjugated recombinant myosin.
[0013]
[Means for Solving the Problems]
This application is directed to Ca as an invention for solving the above-mentioned problems. ²⁺ Bound myosin heavy chain, Ca ²⁺ An expression product of a polynucleotide encoding each of a bound light chain and a phosphorylated light chain, wherein wild-type Ca ²⁺ Provided is a recombinant myosin having substantially the same function as conjugated myosin.
[0014]
Another embodiment of the present invention is a myosin heavy chain, Ca ²⁺ One or more amino acid residues in the amino acid sequence of the combined light chain and / or phosphorylated light chain are replaced with other amino acid residues, one or more amino acid residues are deleted, or one or more amino acid residues are added Recombinant myosin.
[0015]
Moreover, in the recombinant myosin of this invention, it is set as a preferable aspect that each polynucleotide is derived from a true slime mold Phisalum.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
The recombinant myosin of the present invention is Ca ²⁺ Bound myosin heavy chain (approximately 220 kDa), Ca ²⁺ Expression products of polynucleotides encoding each of the bound light chain (about 16 kDa) and the phosphorylated light chain (about 18 kDa).
[0017]
"Ca ²⁺ `` Binding myosin '' means Ca <sup>2+</sup> Specifically, for example, myosin of true slime mold Physarum (saruopu) and scallop (scallop). In addition, "wild type Ca" <sup>2+</sup> Specifically, `` having substantially the same function as bound myosin '' means that myosin decomposes ATP and binds to actin to generate mechanical energy. ²⁺ Means giving change. However, recombinant myosin derived from physalum is not Ca ²⁺ Binding reduces ATP degradation and the generation of mechanical energy, scallop-derived recombinant myosin is Ca ²⁺ It means that the binding increases ATP degradation and generation of mechanical energy.
[0018]
The recombinant myosin of the present invention can be prepared by expressing the above polynucleotide in an in vitro translation system or an appropriate host-vector system.
[0019]
For example, polynucleotide (cDNA) is known base sequence information (eg, myosin heavy chain of physalum: GenBank No. AF335500, Ca ²⁺ Light chain: GenBank No. J03499, phosphorylated light chain: GenBank No. AB076705; scallop myosin heavy chain: GenBank No. X55714, light chain: GenBank No. M17208, M17201) Can be obtained by screening a cDNA library. The target polynucleotide can also be obtained by PCR or RT-PCR using oligonucleotide primers prepared based on known nucleotide sequence information.
[0020]
The obtained polynucleotides can be separately cloned into an expression vector and co-expressed to produce the recombinant myosin of this invention. Alternatively, it may be cloned into an expression vector as a fusion polynucleotide in which each polynucleotide is linked. However, in that case, heavy chain, Ca ²⁺ A stop codon is provided at the 3 ′ end of each polynucleotide so that the bound light chain and the phosphorylated light chain are each expressed as a mature protein. Each polynucleotide may be provided with a respective expression control sequence (promoter / enhancer) at the 5 ′ end of each polynucleotide.
[0021]
When expressing the recombinant myosin of the present invention in an in vitro translation system, a polynucleotide is recombined into an expression vector having an RNA polymerase promoter, and the rabbit reticulocyte lysate containing this recombinant vector and an RNA polymerase corresponding to the promoter And in vitro translation systems such as wheat germ extract. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Examples of vectors containing these RNA polymerase promoters include pKA1, pCDM8, pT3 / T718, pT7 / 319, and pBluescript II.
[0022]
If the polynucleotide is expressed in an appropriate host-vector system, recombinant myosin can be produced in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, mammalian cells, and plant cells. can do. For example, when expressed in a microorganism such as Escherichia coli, an expression vector is prepared by recombining a polynucleotide with an expression vector having an origin, promoter, ribosome binding site, DNA cloning site, terminator, etc. that can replicate in the microorganism, If host cells are transformed with this expression vector and the transformant is cultured, the desired recombinant myosin can be mass-produced from the culture. Examples of the expression vector for E. coli include pUC system, pBluescript II, pET expression system, and pGEX expression system. Furthermore, when expressing recombinant myosin in eukaryotic cells, a polynucleotide is inserted into an expression vector for eukaryotic cells having a promoter, a splicing region, a poly (A) addition site, etc. to produce a recombinant vector, The desired recombinant myosin can be obtained from eukaryotic cells transfected with this vector. Examples of expression vectors include pKA1, pCDM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYES2. As eukaryotic cells, mammalian cultured cells such as human fetal kidney cells HEK293, monkey kidney cells COS7, Chinese hamster ovary cells CHO, or primary cultured cells isolated from human organs can be used. Budding yeast, fission yeast, silkworm cells, Xenopus egg cells and the like can also be used. In addition, when the above expression vector is transfected into insect cells together with the genomic DNA of a nuclear polyhedrosis virus belonging to the Baculoviridae family, the desired recombinant myosin can be obtained from the insect cells. As insect cells, Sf9, Sf21, Tn5, etc. can be used.
[0023]
In order to introduce the expression vector into cells, known methods such as electroporation, calcium phosphate method, liposome method, DEAE dextran method and the like can be used. Isolation and purification of the polypeptide expressed in the transformed cell can be performed by combining known separation operations. For example, treatment with denaturing agents and surfactants such as urea, ultrasonic treatment, enzyme digestion, salting out and solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.
[0024]
Another aspect of the invention is a myosin heavy chain, Ca ²⁺ One or more amino acid residues in the amino acid sequence of the combined light chain and / or phosphorylated light chain are replaced with other amino acid residues, one or more amino acid residues are deleted, or one or more amino acid residues are added Is a modified recombinant myosin.
[0025]
The “modified type” in this case means that the activity of the recombinant myosin (for example, Ca2 + binding ability, ATP resolution, mechanical energy generation ability, etc.) is increased or decreased by the mutation of the amino acid sequence. Alternatively, it means an amino acid mutation that increases or decreases the binding properties with other molecules and compounds.
[0026]
Such an amino acid mutation can be performed by introducing a mutation into the 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 substituted with another amino acid residual codon, the known Kunkel method (Kunkel, TA Proc. Natl. Acad. Sci. USA 82: 488, 1985 and Kunkel, TA , et al. Methods in Enzymology 154: 367, 1987). Ie dut ^- , Ung ^- Escherichia coli (BW313, CJ236, etc.) indicated by the genotype of is dUTPase (Dut) and Uracil-DNA glycosylase (Ung) deficient, so a portion of thiamine (T) in DNA is deoxyuracil (dU) Synthesize DNA that has been replaced with. Using this Escherichia coli as a host fungus, an oligonucleotide designed to substitute the target residue with another amino acid residue is hybridized in vitro with the polynucleotide targeted for mutagenesis. A complementary DNA strand is synthesized by a ligase reaction. Ung this DNA ⁺ When introduced into E. coli strains (DH5α, etc.), the original DNA strand containing dU is degraded by Ung, but the complementary DNA strand synthesized in a test tube is replicated without degradation. In this way, the DNA strand on which the mutation is introduced is selectively amplified, and a polynucleotide encoding myosin containing the target mutation can be obtained. Mutant polynucleotides can also be obtained by methods such as using mutation kits, mutagenesis-type PCR methods, or known polynucleotide synthesis methods (for example, Nucleic Acid Res. 25: 3440-3444, 1997). Obtainable.
[0027]
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 by the following examples.
[0028]
【Example】
(1) Materials and methods
(1-1) Chemical substances
Restriction enzymes and other enzymes were from Takara Shuzo Co., Ltd. (Kyoto). All other reagents used 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 sub-fragment-1 heavy chain (S1-HC; Met1-Gly841) of physalum-derived myosin heavy chain was synthesized by RT-PCR (KOD DNA polymerase; Toyobo Co., Ltd., Tokyo). The following PCR primers designed based on known sequence information (GenBank No. AF335500) were used.
[0029]
5'-CGGGATCCATGGCAAGCGAAAGGCAAC-3 '(SEQ ID NO: 1)
5'-ATGGTGCTTGTCGTCGTCGTCGCCAACCAATAAGGGACGCG-3 '(SEQ ID NO: 2)
PCR conditions were denaturation (94 ° C. for 1 minute), annealing (55 ° C. for 1 minute), and extension (72 ° C. for 3 minutes) as one cycle, and 35 cycles were performed.
[0030]
In addition, in order to express recombinant myosin, a hexa-His-tag sequence was attached to the C-terminus by PCR. The following primers at the end of S1-HC:
5'-CCGCGGCCGCATGATGATGATGATGGTGCTTGTCGTCGTCGTCGTC-3 '(SEQ ID NO: 3)
Contained a hexa-His tag sequence, a stop codon and a SalI restriction site.
Nest PCR was performed using the following primers.
[0031]
5'-CGGGATCCATGGCAAGCGAAAGGCAAC-3 '(SEQ ID NO: 4)
The PCR product obtained above was cloned into the BamHI / SalI site of pBlueBac4.5a (Invitrogen, Carlsbad, CA, USA), and the DNA sequence was confirmed. The obtained plasmid was designated as pBB / S1.
[0032]
An introduction vector for the heavy meromyosin (HMM) heavy chain (Met1-Lys1181) of the myosin heavy chain was also constructed in the same manner as described above. A coiled-coil structural region, which is a part of the myosin heavy chain, was added to the C-terminus of S1-HC by PCR. Primer with SmaI restriction site (2044 bp of myosin heavy chain):
5′-CAGAAGCCCGGGTACCTTG-3 ′ (SEQ ID NO: 5),
Primer containing HMM fragment (Lys1181) at the end:
5'-ATGATGATGGTGCTTGAGCTCCTCTACCTG-3 '(SEQ ID NO: 6)
Was used. Primer to give the hexa-His tag sequence:
5′-CAGAAGCCCGGGTACCTTG-3 ′ (SEQ ID NO: 7);
Primer containing hexa-His tag sequence, stop codon, SalI restriction site:
5'-GGACTAGTGTCGACTTAATGATGATGATGATGGTGC-3 '(SEQ ID NO: 8)
Nest PCR was performed using.
[0033]
The obtained PCR product was cloned into SmaI / SalI of the pBB / S1 plasmid, and the plasmid was constructed as pBB / HMM.
[0034]
Furthermore, using primers designed based on the known sequence information, introduction vectors for polynucleotides encoding Ca2 + -binding light chain (CaLC) and phosphorylated light chain (PLC) were constructed. BamHI or KpnI restriction enzyme sites were added to the 5 ′ ends of PLC and CaLC. In addition, a KpnI or EcoRI site was added to the 3 ′ end of PLC and CaLC. The obtained PCR products were subcloned into the BamHI / KpnI and KpnI / EcoRI restriction sites of pBlueBac4.5a, respectively, to construct plasmids pBB / PLC and pBB / CaLC.
(1-3) Recombinant virus infection and selection
Spodoptera frugiperda (Sf-9) cells 75cm ² Incubated at 27 ° C. For the culture solution (TNM-FH), 10% fetal calf serum (FCS) and 10 μg / ml gentamicin (Sigma-Aldrich, USA) were added to Grace's insect cell culture solution (Invitrogen, Carlsbad, CA, USA). In Sf-9 cells, linear DNA (Bac-M-Blue) of the nuclear multifaceted disease virus Auto-graphica californica ^TM DNA; Invitrogen, Carlsbad, CA, USA) and one of the transfer vectors pBB / S1, pBB / HMM, pBB / PLC and pBB / CaLC. InsectinPlus to increase the efficiency of infection introduction ^TM Liposome reagent (Invitrogen, Carlsbad, CA, USA) was used. Seven days later, a plaque assay was performed on X-gal containing plates to isolate recombinant baculovirus. Pick up the blue plaque, Sf-9 cells (25cm ² The recombinant baculovirus was amplified. After 4 days, the recombinant baculovirus in the supernatant was ² Stocks with high virus titers were prepared by infecting Sf-9 cells cultured for 11 days in flasks. Various introduced cDNAs incorporated into the recombinant baculovirus were confirmed by PCR and subsequently also confirmed by a nucleotide sequence analyzer. The resulting recombinant baculovirus was again propagated by the method described above.
(1-4) Purification of recombinant S1 and HMM
72cm ² 1 x 10 in each of the 10 culture vessels ⁷ Sf-9 cells were seeded. Sf-9 cells contain S1-heavy or HMM-heavy chain, phosphorylated light chain and Ca ²⁺ Each baculovirus introduced with a polynucleotide encoding each bound light chain was co-infected individually. In all cases, the multiplicity of infection (moi) was 5. Infected Sf-9 cells were grown at 27 ° C. for 3 days, and then centrifuged at 4 ° C. for 10 minutes at a speed of 1500 rpm to collect the cells. Since the collected cells are in pellet form, 7 ml homogenization buffer [20 mM Tris-HCl (pH 7.5), 1 mM MgCl ₂ , 1 mM EGTA, 5 mM 2-mercaptoethanol, protease inhibitor 1 mM p-ABSF (Wako Pure Chemical Industries, Ltd.), 10 μg / ml leupeptin and His tag protease inhibitor (Sigma-Aldrich, USA) )] And then homogenized. After homogenization, adjust to 0.2M NaCl and 1mM ATP to release recombinant myosin from endogenous actin, and centrifuge for 15 minutes at 15000 xg to remove cell debris and unbroken cells Went. The supernatant was mixed with 0.2 mg / ml rabbit skeletal muscle actin (purified from rabbit skeletal muscle acetone powder) and buffered (20 mM Tris-HCl (pH 7.5), 50 mM KCl, 10 mM MgCl ₂ , 0.3 mM DTT, 1 mM p-ABSF and 1 μg / ml leupeptin) for 24 hours. Actin recombinant HMM was formed and precipitated during dialysis and collected by centrifugation at 100000 × g for 1 hour. The S1 / HMM precipitate separated from the precipitate was added to the buffer [20 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl ₂ , 7 mM 2-mercaptoethanol, 100 μM p-ABSF, 1 μg / ml leupeptin and 1 mM ATP], centrifuged at 100,000 × g for 90 minutes, and the resulting supernatant was treated with Ni-NTA spur flow (Qiagen In Germany). His-tagged recombinant S1 or HMM was trapped in this column and extracted with an extract [20 mM Tris-HCl (pH 7.5), 40 mM KCl, 7 mM 2-mercaptoethanol, 1 μg / ml leupeptin and 100 mM imidazole]. After performing SDS-PAGE, the portion of the electrophoresis band containing recombinant S1 / HMM was accumulated and concentrated using a Centriprep-30 concentrator (Millipore, USA). Next, recombinant myosin was buffered [20 mM Tris-HCl (pH 7.5), 40 mM KCl, 1.5 mM MgCl]. ₂ , 0.1 mM DTT, 20 μM p-ABSF and 0.6 μg / ml leupeptin]. Protein purification was performed at a temperature of 4 ° C. or lower. 0.1 mg of typical recombinant S1 / HMM was collected and quantified with Bio-Rad protein measurement solution using BSA as a standard value. ⁸ Sf-9 cells were obtained.
(1-5) Gel electrophoresis
SDS-PAGE uses a 12.5% 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). I went. The SDS-sample buffer consisted of 40 mM Tris-HCl (pH 6.8), 50 mM DTT, 1% SDS, 7.5% glycerol, 0.002% bromophenol blue.
(1-6) Observation with an electron microscope
Purified HMM (0.5 mg / ml) was placed on a mica sheet, which was washed three times with 0.1 M ammonium acetate containing 30% glycerol, stained with 2% uranyl acetate aqueous solution, and washed three times. This mica sheet is covered with another divided mica sheet, pressed, peeled off, and then replicated on platinum at a low angle in a BAF 060 rotary imaging system. Observed with.
(1-7) Analysis by ATPase activity
Measurement of ATPase activity in S1 / HMM was performed according to the description in the literature (Anal Biochem. 293: 212-215, 2001). All analyzes were performed at 25 ° C. Basic Mg ²⁺ -ATPase activity was analyzed with 20 mM Tris-HCl (pH 7.5), 40 mM KCl, 1.5 mM MgCl2, 0.1 mM DTT, 0.5 mM ATP, polymerized actin derived from rabbit skeletal muscle, and 0.5 μM recombinant S1 / HMM It was. Statistical analysis was performed using Student's t-test. <0.05 was considered statistically significant.
(1-8) Ca ²⁺ Bond measurement
Ca ²⁺ The binding range of is 3.5 μM HMM, 50 μM recombinant Ca as described in the literature (Biochemistry 39: 3827-3834, 2000). ²⁺ CaCl in the presence of light chain or 50 μM recombinant phosphorylated light chain ₂ 0.1 mM NaCl, 5 mM MgCl containing (Du-Pont-NEN) ₂ And it measured at 25 degreeC using the fluid dialysis method using MOPS / NaOH (pH 7.0). Phosphorylated light chain or Ca ²⁺ An NdeI restriction enzyme site was added to the 5 ′ end of the ORF of each light chain, and a BamHI site was added to the 3 ′ end by PCR. This PCR product was treated with NdeI and BamHI and inserted into the same site of the pET19b expression vector system (Novagen, USA). According to the manufacturer's instructions, pET19b / PLC or pET19b / CaLC was expressed and purified in E. coli BL21 (DE3) strain. All data were analyzed by fitting to the Adiar equation.
(1-9) Motor force analysis in In Vitro
The analysis of motor force in In Vitro was performed according to the description 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) is labeled with rhodamine phalloidin (Molecular Probe, USA), and a motion analysis medium [10 mM KCl, 2 mM ATP, 1 mM MgCl ₂ , 10 mM imidazole (pH 7.5), 14 mM 2-mercaptoethanol and 0.1 mM EGTA or 0.1 mM CaCl ₂ I put it on top. ATP-dependent movements were observed with a fluorescence microscope and recorded with a video camera. The speed of the actin movement was calculated from the distance moved and the time elapsed by capturing the movement with a video camera. Statistical analysis was performed using Student's t-test. <0.05 was considered statistically significant.
(2) Results
Sf-9 cells were infected with S1-heavy chain recombinant baculovirus. After 3 days of culture, these cells were collected, homogenized, and centrifuged. The expression product is in the precipitate (FIG. 1), indicating that the S1-heavy chain is insoluble. Similarly, S1-heavy chain baculovirus and Ca ²⁺ The expression product was obtained as an insoluble material upon co-infection of the bound light chain with baculovirus. However, this S1-heavy chain recombinant baculovirus and phosphorylated light chain recombinant baculovirus and Ca ²⁺ Co-infection of the bound light chain with recombinant baculovirus produced soluble S1-heavy chain in Sf-9 cells (FIG. 2). Similarly for HMM-heavy chain, phosphorylated light chain and Ca ²⁺ Soluble product was obtained when co-infection of the bound light chain with recombinant baculovirus (FIG. 3).
[0035]
S1-heavy chain, phosphorylated light chain and Ca ²⁺ To recover soluble S1-heavy chains from raw extracts of Sf-9 cells infected with recombinant baculovirus with bound light chain, a large amount of actin was mixed with this extract and centrifuged . The resulting pellet was suspended in a buffer containing ATP, and the S1-heavy chain was released from the pellet. The remaining actin was removed by centrifugation again, and the resulting supernatant was injected into a Ni-NTA spar flow column. As shown in FIG. 2, the extracts were 98 kDa (S1-heavy chain), 18 kDa (phosphorylated light chain), 16 kDa (Ca ²⁺ It is composed of three large bands (binding light chain). Thereby, it can be seen that the extract contains S1. HMM consists of HMM-heavy chain peptide 135 kDa, phosphorylated light chain peptide 18 kDa and Ca ²⁺ Similar to the peptide 16 kDa complex of bound light chain (FIG. 3). A double-headed HMM was shown by observation with an electron microscope (FIG. 4). The ATPase activity of S1 and HMM was analyzed by high performance liquid chromatography that can be quantified even with a small amount of sample. In the recombinant S1, ATPase activity was confirmed, and this activity was activated by actin as follows. That is, 0.12 ± 0.01 (s ^-1 head ^-1 ) (N = 3) (m ± SEM) Mg ²⁺ -ATPase activity (Fig. 5); and in the presence of various concentrations of actin, the activity is K _m V with 2.5μM _max = 0.61 (s ^-1 head ^-1 (Fig. 6). Thus, it was confirmed that S1 was activated up to about 5 times by actin and functional S1 was expressed. Similarly for HMM, 0.21 ± 0.02 (s ^-1 head ^-1 ) (N = 3) (m ± SEM) Mg ²⁺ -ATPase activity was shown (FIG. 7). Actin is K _m = 1.8 μM activity by V _max = 1.27 (s ^-1 head ^-1 (FIG. 8), and it was confirmed that HMM was activated up to about 6 times by actin.
[0036]
Ca in ATPase activity of S1 ²⁺ The effect of was tested under maximal activation conditions with actin. Ca ²⁺ In the presence of EGTA, the chelating agent, the activity is 0.49 ± 0.07 (s ^-1 head ^-1 ) (N = 3). 0.1 mM Ca ²⁺ The activity is 0.45 ± 0.04 (s ^-1 head ^-1 ) (N = 3) slightly decreased (FIG. 5). However, this decrease was not statistically significant. Ca ²⁺ The effect of was tested in the same way with HMM. However, the effect was minimal, if any (Figure 7).
[0037]
Ca ²⁺ Since S1 and HMM were not detected ²⁺ The question of whether or not to join is raised. Therefore, Ca by HMM ²⁺ Binding was confirmed by flow dialysis. As shown in FIG. 9, the phosphorylated light chain is Ca ²⁺ Did not combine with. But Ca ²⁺ Bound light chain is Ca ²⁺ Binding activity was confirmed. The binding activity of HMM was dramatically increased; the maximum binding activity was K versus 2 mol / mol HMM. _d Was at the 10 μM level.
[0038]
Ca against recombinant HMM ²⁺ The effect of binding was examined using in vitro kinetic analysis. That is, ATP-dependent movement of actin was observed on the glass surface coated with HMM (FIG. 10). The average velocity in the presence of EGTA is 0.61 ± 0.16μm / sec (n = 25), while Ca ²⁺ In the presence, it decreased to 0.32 ± 0.21 μm / sec (n = 25). From this result, the recombinant HMM ²⁺ Having binding activity and Ca ²⁺ Was confirmed to act on the motor function activity of recombinant HMM.
[0039]
【The invention's effect】
As explained in detail above, according to the invention of this application, Ca ²⁺ A conjugated recombinant myosin is provided. This recombinant myosin is useful as an actuator in a micromachine, a switch element of an electronic circuit, or the like.
[0040]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the results of SDS-PAGE of recombinant S1-heavy chain expressed in Sf-9 cells. Lane 1 is a molecular weight marker, lane 2 is a homogenate of uninfected Sf-9 cells, lane 3 is a precipitate of infected Sf-9 cell homogenate, and lane 4 is a supernatant of infected Sf-9 cell homogenate.
FIG. 2 shows the result of SDS-PAGE during the S1 purification process. Lane 1 is the separation marker, Lane 2 is the homogenized and centrifuged supernatant of uninfected Sf-9 cells, Lane 3 is the same cell precipitate, Lane 4 is the S1-heavy chain, phosphorylated light chain and Ca. ²⁺ Sf-9 cells infected with the bound light chain were homogenized and centrifuged, lane 5 was the same cell precipitate, lane 6 was purified from a complex of S1 and actin, lane 7 was Ni- S1 purified by NTN column.
FIG. 3 shows the results of SDS-PAGE during the purification process of recombinant HMM. Lane 1 is molecular weight marker, lane 2 is the supernatant of uninfected Sf-9 cell homogenate, lane 3 is its precipitate, lane 4 is S1-heavy chain, phosphorylated light chain and Ca ²⁺ The supernatant of the Sf-9 cell homogenate infected with the bound light chain, 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. .
4 is an electron micrograph of the recombinant HMM purified in the process of FIG.
FIG. 5 is a measurement result of S1 ATPase activity. 1 is the reference Mg in the presence of 0.1 mM EGTA ²⁺ -ATPase activity, 2 is actin-activated ATPase activity in the presence of 0.1 mM EGTA and 5 μM actin, 3 is 0.1 mM Ca ²⁺ Actin-activated ATPase activity in the presence. Values are mean ± SEM of 3 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 fixed at 0.5 μM and 0.1 mM, respectively. Other measurement conditions were the same as in FIG.
FIG. 7 is a measurement result of ATPase activity of recombinant HMM. 1 is the reference Mg in the presence of 0.1 mM EGTA ²⁺ -ATPase activity, 2 is actin-activated ATPase activity in the presence of 0.1 mM EGTA and 5 μM actin, 3 is 0.1 mM Ca ²⁺ And actin-activated ATPase activity in the presence of 5 μM actin. Values are mean ± SEM of 3 measurements.
FIG. 8 shows measurement results of ATPase activity of recombinant HMM in the presence of various concentrations of actin.
FIG. 9 is a measurement result of calcium binding activity of recombinant HMM. The black circle is HMM, the white circle is CaLC, and the black diamond is PLC.
FIG. 10 shows Ca against HMM motor activity. ²⁺ It is the result of measuring the effect of. A is the measurement result in the presence of 0.1 mM EGTA, B is 0.1 mM Ca ²⁺ It is a measurement result in the presence. The arrow indicates the average rate of ATP-dependent movement of actin.

Claims (1)

Intrinsic slime mold Fizarumu derived Ca ²⁺ binding type myosin heavy chain subfragments -1 heavy chain, an expression product of a polynucleotide encoding Ca ²⁺ binding light chain, and each of phosphorylated light chain, Ca ^{2 +} Recombinant myosin characterized by reducing ATP degradation and generation of mechanical energy by binding .

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