purification of myosin-7a using flag-affinity chromatography

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

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.

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.

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.

purification-of-myosin-7a-using-flag-affinity-chromatography

Research

JoVE Journal

Biochemistry

10 次观看. 首先，准备 master buffer。使用 0.22 微米孔径过滤器过滤，并将缓冲液储存在 4 摄氏度。接下来，通过在主缓冲液中补充三种不含 EDTA 的蛋白酶抑制剂片剂、2 毫摩尔 ATP 和 0.1 毫摩尔 DTT，制备 75 毫升提取缓冲液。然后将 75 毫升提取缓冲液添加到表达肌球蛋白 VIIA 的冷冻细菌细胞沉淀中，并将沉淀物留在冰上的提取缓冲液中直至解冻。之后，使用?...