<meta charset="utf8"></head><body>Protocolo de Producción y Caracterización Funcional de Miosina-7a Humana Recombinante

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL microcentrifuge tubes</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>1X FLAG Peptide</td>
			<td>GenScript</td>
			<td>N/A</td>
			<td>Custom peptide synthesis</td>
		</tr>
		<tr>
			<td>22x22mm No. 1.5 coverslips</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Conical Centrifuge Tubes</td>
			<td>Nunc</td>
			<td>376814</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Vented Erlenmyer Shaker Flask</td>
			<td>IntelixBio</td>
			<td>DBJ-SF250VP</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>VWR</td>
			<td>M131</td>
			<td></td>
		</tr>
		<tr>
			<td>75x25x1 mm Vistavision microscope slides</td>
			<td>VWR</td>
			<td>16004-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin Protein (&#62;99% Pure)</td>
			<td>Cytoskeleton</td>
			<td>AKL99</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 Centrifugal Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 Centrifugal Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>UFC801024</td>
			<td></td>
		</tr>
		<tr>
			<td>ANTI-FLAG M2 Affinity Gel</td>
			<td>Millipore Sigma</td>
			<td>A2220</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Millipore Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Millipore Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Spin Disposable Chromatography Column</td>
			<td>Bio-Rad</td>
			<td>732-6008</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2530H</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluo-Gal</td>
			<td>Thermo Fisher</td>
			<td>15519028</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>BstXI Enzyme</td>
			<td>New England Biolabs</td>
			<td>R0113S</td>
			<td></td>
		</tr>
		<tr>
			<td>Calmodulin</td>
			<td>Millipore Sigma</td>
			<td>208694</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Millipore Sigma</td>
			<td>C40</td>
			<td></td>
		</tr>
		<tr>
			<td>Champion pET-SUMO Expression System</td>
			<td>Thermo Fisher</td>
			<td>K30001</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche Diagnostics</td>
			<td>5056489001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cutsmart Buffer</td>
			<td>New England Biolabs</td>
			<td>B6004S</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Millipore Sigma</td>
			<td>DO632</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Millipore Sigma</td>
			<td>DO632</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I, Spectrum Chemical</td>
			<td>Fisher Scientific</td>
			<td>18-610-304</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Sided Tape</td>
			<td>Office Depot</td>
			<td>909955</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA, Molecular Biology Grade</td>
			<td>Millipore Sigma</td>
			<td>324626-25GM</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA, Molecular Biology Grade</td>
			<td>Millipore Sigma</td>
			<td>324626-25GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Thermo Fisher</td>
			<td>BP2818</td>
			<td></td>
		</tr>
		<tr>
			<td>ExpiFectamine Sf Transfection Reagent</td>
			<td>Gibco</td>
			<td>A38915</td>
			<td></td>
		</tr>
		<tr>
			<td>FAST program</td>
			<td>http://spudlab.stanford.edu/fast-for-automatic-motility-measurements;&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Model 505 Sonic Dismembrator</td>
			<td>Fisher Scientific</td>
			<td>FB505110</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin Reagent Solution</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td>10 mg/mL in distilled water</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Millipore Sigma</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose Oxidase</td>
			<td>Millipore Sigma</td>
			<td>G2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>HisPur Cobalt Resin</td>
			<td>Thermo Fisher</td>
			<td>89966</td>
			<td></td>
		</tr>
		<tr>
			<td>I-CeuI Enzyme</td>
			<td>New England Biolabs</td>
			<td>R0699S</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Stabilizer Plugin</td>
			<td>https://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ FIJI</td>
			<td>https://imagej.net/Fiji/Downloads</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Millipore Sigma</td>
			<td>I2399</td>
			<td></td>
		</tr>
		<tr>
			<td>In-Fusion Snap Assembly Master Mix</td>
			<td>TaKaRa</td>
			<td>638948</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Thermo Fisher</td>
			<td>15529019</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A451SK</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientific</td>
			<td>AAJ67354AD</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Orifice Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-134</td>
			<td>1-200uL</td>
		</tr>
		<tr>
			<td>LB Agar, Ready-Made Powder</td>
			<td>Thermo Fisher</td>
			<td>J75851-A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Leupeptin Protease Inhibitor</td>
			<td>Thermo Fisher</td>
			<td>78435</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Thermo Fisher</td>
			<td>J61014.=E</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Thermo Fisher</td>
			<td>J61014.=E</td>
			<td>1M</td>
		</tr>
		<tr>
			<td>Max Efficiency DH10Bac Competent Cells</td>
			<td>Gibco</td>
			<td>10361012</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, 1.7mL</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, 1.7mL</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Tubes, 1.7mL</td>
			<td>VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>Model: Eclipse Ti with H-TIRF system with 100X TIRF objective</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>ORCA-Fusion BT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Laser Unit</td>
			<td>Andor iXon Ultra</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Miller&#39;s LB Broth</td>
			<td>Corning</td>
			<td>46-050-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Millipore Sigma</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Millipore Sigma</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB 5-alpha Competent E.coli (High Efficiency)</td>
			<td>New England Biolabs</td>
			<td>C2987H</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer r3.1</td>
			<td>New England Biolabs</td>
			<td>B6003S</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose</td>
			<td>LADD Research Industries</td>
			<td>53152</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>pACEBac1 Vector</td>
			<td>Geneva Biotech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Millipore Sigma</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Millipore Sigma</td>
			<td>78830</td>
			<td></td>
		</tr>
		<tr>
			<td>PureLink RNase A (20 mg/mL)</td>
			<td>Invitrogen</td>
			<td>12091021</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (50)</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit (50)</td>
			<td>QIAGEN</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick CIP</td>
			<td>New England Biolabs</td>
			<td>M0525S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine phalloidin</td>
			<td>Invitrogen</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C. Medium</td>
			<td>Invitrogen</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>SENP2 protease</td>
			<td></td>
			<td>PMID:17591783</td>
			<td>Purified in the lab</td>
		</tr>
		<tr>
			<td>Sf9 cells</td>
			<td>Thermo Fisher</td>
			<td>11496015</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf-900 III SFM (1X) - Serum Free Media Complete</td>
			<td>Gibco</td>
			<td>12658-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 3 mL</td>
			<td>Thermo Fisher</td>
			<td>A52971</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Millipore Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup Quick Release Vacuum Driven Disposable Filtration System</td>
			<td>Millipore Sigma</td>
			<td>S2GPU01RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 75 Increase 10/300 GL</td>
			<td>Cytiva</td>
			<td>29148721</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase Buffer - 10X with 10mM ATP</td>
			<td>New England Biolabs</td>
			<td>B0202A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline Hydrochloride</td>
			<td>Millipore Sigma</td>
			<td>T7660-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Millipore Sigma</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X</td>
			<td>American Bioanalytical</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

multibac system-based purification and biophysical characterization of human myosin-7a

Myosins are molecular motor proteins that interact with actin to drive numerous cellular processes1,2,3,4. Humans possess 12 classes and 39 myosin genes5, which are involved in a wide range of physiological functions, such as muscle contraction6 and sensory processes7. Each myosin molecule is a multimeric complex composed of a heavy chain and light chains. The heavy chain is divided into head, neck, and tail regions. The head contains actin- and nucleotide-binding sites that are responsible for ATP hydrolysis and generating force on actin filaments2. The neck is formed by several &#945;-helical IQ motifs where a specific set of light chains are bound. They together function as a lever arm to amplify the motor&#39;s conformational changes into large movements8,9,10. The tail contains class-specific subdomains and plays a regulatory role in tuning myosin&#39;s motor activity and mediating interactions with cellular binding partners2,11.
Human myosin-7a, a member of class-7 myosins, is essential for auditory and visual processes12,13. The IQ motifs of human myosin-7a are associated with a unique combination of light chains, including the regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4)14,15,16. Besides stabilizing the lever arm, these light chains regulate the mechanical properties of myosin-7a in response to calcium signaling, a feature that appears to be unique to the mammalian isoform14.
Defects in the gene encoding the myosin-7a heavy chain (MYO7A/USH1B) are responsible for Usher syndrome type 1, the most severe form of combined vision and hearing loss in humans17. Additionally, the light chain gene CALML4, is among the candidate genes mapped to contain the causative allele for USH1H, another variant of type 1 Usher syndrome15,18. In the retina, myosin-7a is expressed in the retinal pigment epithelium and photoreceptor cells13. It has been implicated in the localization of melanosomes in the retinal pigment epithelium (RPE)19 and phagocytosis of photoreceptor outer segment disks by the RPE cells20. In the inner ear, myosin-7a is primarily found in the stereocilia, where it plays a critical role in establishing hair bundles and in gating the mechano-electrical transduction process12,21,22.
While the importance of myosin-7a in sensory cells is well established, its functional mechanisms at the molecular level remain poorly understood. This gap in knowledge is partly due to the challenges in purifying the intact protein, especially the mammalian isoform. Recently, significant progress has been made using the MultiBac system to recombinantly express the complete human myosin-7a holoenzyme14. This advancement has enabled structural and biophysical characterizations of this motor protein, leading to the discovery of several unique properties of human myosin-7a that are specifically adapted for mammalian auditory functions14,23.
The MultiBac system is an advanced baculovirus/insect cell platform specifically designed for the expression of eukaryotic multimeric complexes24,25. A key feature of this system is its ability to host multiple gene expression cassettes, each encoding a subunit of the complex, within a single MultiBac baculovirus. The assembly of the multigene expression cassettes is facilitated through a so-called multiplication module: a homing endonuclease (HE) site and a matching designed BstXI site flanking the multiple cloning sites (MCS). This module enables the iterative assembly of a single expression cassette by restriction/ligation, leveraging the fact that the HE and BstXI restriction sites are eliminated upon their ligation. In this paper, human myosin-7a heavy chain, RLC, calmodulin, and CALML4 are each cloned into the multiplication module within the pACEBac1 vector (Figure 1A), which are then assembled into a multigene expression cassette through the iterative process (Figure 1B). The myosin-7a multigene cassette is integrated into the baculoviral genome (bacmid) through the transposition of the mini-Tn7 element from the pACEBac1 vector to the mini-attTn7 target site in the genome (Figure 1C). Following procedures for bacmid purification, baculovirus production, and amplification (Figure 1D,E), the recombinant myosin-7a MultiBac baculovirus is prepared and can be used for large-scale protein production (Figure 1F). Additionally, the myosin-7a light chains can be produced separately in E. coli and purified using a cleavable His6-SUMO tag26,27,28. The purified light chains are useful for studying their binding dynamics and regulation of myosin-7a.
The purified myosin-7a protein can be subjected to structural, biochemical, and biophysical studies to gain insights into the structural-functional regulation of this motor protein. Additionally, its interactions with the actin network and other binding proteins29 can be examined using a variety of in vitro reconstitution approaches. Findings from these analyses will inform the biophysical properties of this myosin, leading to a mechanistic understanding of how myosin-7a drives the cytoskeletal changes and ultimately shapes the unique morphology and function of sensory cells. In this paper, we detail a workflow for actin filament gliding assay that has been specifically adapted for mammalian myosin-7a. Actin filament gliding assay is a robust in vitro motility assay that quantitatively studies the movement of fluorescent actin filaments propelled by a large number of myosin motors immobilized on a coverslip surface30,31,32. The advantages of this assay include its simplicity of setup, minimal equipment requirements (a wide-field fluorescence microscope equipped with a digital camera), and high reproducibility. Additionally, because the motion of actin filaments is driven by a cluster of immobilized myosin motors, this assay is particularly useful for studying the motility of monomeric myosins such as myosin-7a14,33. The protocols include several modifications, from experimental procedures to imaging analysis, specifically tailored to the unique motile properties of mammalian myosin-7a. With the availability of intact myosin-7a protein and the functional characterization protocol outlined here, this paper lays the groundwork for further investigation into the molecular roles of myosin-7a in both physiological and pathological processes.

<meta charset="utf8"></head><body>Autor destacado: Desentrañando el papel de la miosina-7a y las proteínas Usher en la audición y las enfermedades humanas

Purificación y caracterización biofísica de miosina-7a humana basada en el sistema MultiBac

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common ...

multibac-system-based-purification-biophysical-characterization-human

Research

JoVE Journal

Biochemistry

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

291 vistas.  West Virginia University. Estamos tratando de responder por qué las mutaciones en los motores de miosina causan enfermedades humanas y qué roles juegan estas proteínas en el proceso auditivo. Nuestra investigación se centra en comprender los mecanismos moleculares de la miosina-7a y cómo funciona con las proteínas Usher para ensamblar y mantener la maquinaria celular necesaria para la audición. La comprensión de los mecanismos estructurales y funcionales de la miosina-7a requi...

Video: Autor destacado: Desentrañando el papel de la miosina-7a y las proteínas Usher en la audición y las enfermedades humanas

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing.

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.