We thank the Microscopy Imaging Facility and Visual Function and Morphology Core at West Virginia University for discussion and help with image analysis. This work is supported by the tenure-track startup funds from West Virginia University School of Medicine to R.L. This work is also supported by National Institute of General Medical Sciences (NIGMS) Visual Sciences Center of Biomedical Research Excellence (Vs-CoBRE) (P20GM144230), and the NIGMS West Virginia Network of Biomedical Research Excellence (WV-INBRE) (P20GM103434).

NOTE: Here we describe a protocol for synthesizing the intact human myosin-7a holoenzyme and characterizing its motility in vitro. This protocol is divided into three sections: first, expressing the human myosin-7a using the MultiBac baculovirus protein expression system; Second, purifying myosin-7a light chains separately using the E.coli His6-SUMO system; and lastly, studying the motility of human myosin-7a using the actin-filament gliding assay.
1. MultiBac system-based myosi...

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

Presented here is a detailed protocol for the production of recombinant human myosin-7a protein from insect cells. Although the Sf9/baculovirus system has been used to produce a variety of myosins40,41,42,43, only recently has the mammalian myosin-7a been successfully purified using the MultiBac baculovirus system14. Mammalian myosin-7a is found to associate with three t...

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.

<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

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.

Author Spotlight: Unraveling the Role of Myosin-7a and Usher Proteins in Hearing and Human Disease

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

We're trying to answer why mutations in myosin motors cause human disease and what roles these proteins play in the hearing process. Our research focuses on understanding the molecular mechanisms of Myosin-7a and how it works with Usher proteins to assemble and maintain the cellular machinery required for hearing. Understanding the structural and functional mechanisms of Myosin-7a requires high-quality intact protein, which has been a longstanding challenge in the field.Recently, we made significant progress and successfully purified the full-length Myosin-7a hollow enzyme, allowing for further investigation of this important protein. With this protein now available, we can investigate key features of this motor including its structure, force generation and its motility. These insights are crucial for understanding the mechanisms of Myosin-7a in hearing and the molecular defects that lead to deafness in human patients.In the future, our lab will leverage our expertise in the multi vac system to recombinantly produce large biomolecular complexes, for example the Usher 1 complex, which is critical for human sensory functions. This approach will enable us to determine their structures and the principles underlying their roles in sensory cells.

The purified myosin-7a complex and light chain proteins can be evaluated by SDS-PAGE gel electrophoresis, as shown in Figure 3. The band above the 200 kDa marker corresponds to the myosin-7a heavy chain (255 kDa). The three bands migrating between the 22 and 14 kDa markers from top to bottom are RLC (20 kDa), calmodulin, and CALML4, respectively. While calmodulin and CALML4 have a similar molecular weight of approximately 17 kDa, the two proteins can be separated using a 16% Tris-Glycine gel...

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.

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

Purification of Myosin-7a Using FLAG-Affinity Chromatography

To begin, prepare master buffer. Filter it using a 0.22 micrometer pore size filter, and store the buffer at 4 degrees Celsius. Next, prepare 75 milliliters of extraction buffer by supplementing the master buffer with three EDTA-free protease inhibitor tablets, 2 millimolar ATP, and 0.1 millimolar DTT.Then add 75 milliliters of extraction buffer to the myosin VIIA-expressing frozen bacterial cell pellet, and leave the pellet in the extraction buffer on ice until it thaws. After that, use a serological pipette to break up the pellet to accelerate the thawing process. Now sonicate the resuspension on ice for 10 minutes at 50%amplitude using a half inch tip with 5 seconds on and 5 seconds off.Centrifuge the total lysate at 33, 746 G for 30 minutes at 4 degrees Celsius. During the centrifugation, wash 3 milliliters of the 50%slurry of anti-FLAG affinity resin with 40 milliliters of PBS to prepare 1.5 milliliters of FLAG resin. Afterward, centrifuge the resin at 800 G for 2 minutes at 4 degrees Celsius.Carefully remove the PBS without disturbing the resin. Then add the supernatant from the lysate centrifugation to the washed resin and incubate with continuous rotation at 4 degrees Celsius for 1 hour. Pellet the resin by centrifuging at 800 G for 2 minutes at 4 degrees Celsius.And remove the supernatant without disturbing the resin. Next, re-suspend the resin in 40 milliliters of master buffer and centrifuge at 800 G for 2 minutes at 4 degrees Celsius. Carefully remove the supernatant without disturbing the resin.Now transfer the washed resin to two polypropylene spin columns. Wash each column one time with 2 milliliters of master buffer. Then centrifuge the column at 1, 400 G for 2 minutes at 4 degrees Celsius to remove the master buffer.To prepare an elution buffer, add 100 micrograms per milliliter of FLAG peptide to the master buffer. Add 300 microliters of elution buffer to the packed resin in each column. Gently mix by pipetting to ensure the resin is completely hydrated by the elution buffer.Then centrifuge the columns at 1, 400 G for 2 minutes at 4 degrees Celsius. Transfer the flow through to a clean micro centrifuge tube and keep it on ice. Now, perform SDS-PAGE with the elution fractions to determine their relative concentrations.While the gel runs, prepare a dialysis buffer with the given composition. Then combine the most concentrated fractions. Transfer the sample to a 10, 000 molecular weight cutoff dialysis cassette and dialyze overnight at 4 degrees Celsius.The next day, carefully unload the sample from the dialysis cassette. Aliquot 15 microliters per PCR tube and drop the tubes into a container of liquid nitrogen for flash freezing. The purified myosin VIIA complex was confirmed by SDS-PAGE, showing a band above the 200 kilo Dalton marker, corresponding to the myosin VIIA heavy chain, and three distinct bands between the 22 and 14 kilo Dalton markers representing RLC calmodulin and calmodulin-like protein 4.

Studying the Motility of Myosin-7a-Tailored Actin Filament Using an In Vitro Gliding Assay

To begin, add one chamber volume of purified human myosin-7a protein in 500 millimolar sodium chloride motility buffer to the flow chamber, and incubate it for five minutes at room temperature. Wash the flow chamber with three chamber volumes of one milligram per milliliter BSA in 150 millimolar sodium chloride motility buffer. After that, draw the liquid by using a clean wipe at the outlet to absorb excess liquid.Then, flow one chamber volume of 10 nanomolar rhodamine-phalloidin-labeled F-actin in 150 millimolar sodium chloride motility buffer through the flow chamber. Monitor the binding of actin filaments to the surface under a fluorescent microscope with a 100x objective. Ensure optimal actin filament density for tracking, with enough filaments in the field of view but sparse enough to prevent overlapping.Next, wash the flow chamber with three chamber volumes of 150 millimolar sodium chloride motility buffer to remove unbound actin filaments and excess rhodamine-phalloidin. To initiate motility, add one chamber volume of the final buffer to the flow chamber. Then, record images on a fluorescence microscope using 561-nanometer excitation to visualize rhodamine-phalloidin-labeled actin.Find an area on the slide with the desired actin density and capture images every 30 seconds for 30 minutes. Next, download and install the Fast program from the GitHub repository. Ensure that it is the Python 3-compatible version with an additional module for ND2 file conversion.Run the Fast program using a tolerance value of 33 to retain only smoothly moving filaments. Afterward, use the Fast program to analyze the newly created TIFF images. Set the x maximum value to 20, 000 nanometers and the y maximum value to 20 nanometers per second to represent the longest filament length and maximum filament velocity.Then, use px to set the pixel size of the acquired image to 65 nanometers. Set minV to 0.1 nanometers per second as the minimum velocity required for filament inclusion in the analysis. To set the maximum distance allowed between frames for filaments to be linked, use maxD, avoiding the linking of separate filaments between frames.Use pt to set the tolerance for fluctuations in filament velocities, including only those filaments with smooth movements. Lastly, use d to indicate the folder containing the TIFF stacks for analysis. The actin filament gliding assay demonstrated myosin-7a's slow motility, with the video playback sped up 500 times to visualize the movement.The velocity distribution of myosin-7a showed a peak below five nanometers per second, confirming its slow motility.

208 Views.  West Virginia University.  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. 

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

Summary