Name: MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a (Video) | JoVE Journal
Uploaded: 2024-08-23T14:30:00.000+00:00
Duration: 9 min 17 s
Description: Learn to purify human Myosin-7a using the MultiBac Baculovirus system and study its motility with in vitro assays. This method enables investigation of hearing mechanisms and deafness-related molecular defects.

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5 minutes

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).

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

<ol><li>Hartman, M. A., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+myosin+superfamily+at+a+glance%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">The myosin superfamily at a glance.</a> J Cell Sci. 125 (Pt 7), 1627-1632 (2012).</li>
<li>Sellers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosins%3A+a+diverse+superfamily%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Myosins: a diverse superfamily.</a> Biochim Biophys Acta. 1496 (1), 3-22 (2000).</li>
<li>Titus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin-driven+intracellular+transport%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Biol%22%5BJournal%5D)">Myosin-driven intracellular transport.</a> Cold Spring Harb Perspect Biol. 10 (3), a021972(2018).</li>
<li>Quintanilla, M. A., Hammer, J. A., Beach, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-muscle+myosin+2+at+a+glance%5BTitle%5D)+AND+%22J+Cell+Sci%22%5BJournal%5D)">Non-muscle myosin 2 at a glance.</a> J Cell Sci. 136 (5), jcs.260890(2023).</li>
<li>Peckham, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coiled+coils+and+SAH+domains+in+cytoskeletal+molecular+motors%5BTitle%5D)+AND+%22Biochem+Soc+Trans%22%5BJournal%5D)">Coiled coils and SAH domains in cytoskeletal molecular motors.</a> Biochem Soc Trans. 39 (5), 1142-1148 (2011).</li>
<li>Batters, C., Veigel, C., Homsher, E., Sellers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((To+understand+muscle+you+must+take+it+apart%5BTitle%5D)+AND+%22Front+Physiol%22%5BJournal%5D)">To understand muscle you must take it apart.</a> Front Physiol. 5, 90(2014).</li>
<li>Friedman, T. B., Sellers, J. R., Avraham, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unconventional+myosins+and+the+genetics+of+hearing+loss%5BTitle%5D)+AND+%22Am+J+Med+Genet%22%5BJournal%5D)">Unconventional myosins and the genetics of hearing loss.</a> Am J Med Genet. 89 (3), 147-157 (1999).</li>
<li>Geeves, M. A., Holmes, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+mechanism+of+muscle+contraction%5BTitle%5D)+AND+%22Adv+Protein+Chem%22%5BJournal%5D)">The molecular mechanism of muscle contraction.</a> Adv Protein Chem. 71, 161-193 (2005).</li>
<li>Batters, C., Veigel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanics+and+activation+of+unconventional+myosins%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">Mechanics and activation of unconventional myosins.</a> Traffic. 17 (8), 860-871 (2016).</li>
<li>Houdusse, A., Sweeney, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+myosin+generates+force+on+actin+filaments%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">How myosin generates force on actin filaments.</a> Trends Biochem Sci. 41 (12), 989-997 (2016).</li>
<li>Odronitz, F., Kollmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drawing+the+tree+of+eukaryotic+life+based+on+the+analysis+of+2%2C269+manually+annotated+myosins+from+328+species%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species.</a> Genome Biol. 8 (9), R196(2007).</li>
<li>Moreland, Z. G., Bird, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+motors+in+sensory+hair+bundle+assembly%5BTitle%5D)+AND+%22Curr+Opin+Cell+Biol%22%5BJournal%5D)">Myosin motors in sensory hair bundle assembly.</a> Curr Opin Cell Biol. 79, 102132(2022).</li>
<li>Williams, D. S., Lopes, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+many+different+cellular+functions+of+MYO7A+in+the+retina%5BTitle%5D)+AND+%22Biochem+Soc+Trans%22%5BJournal%5D)">The many different cellular functions of MYO7A in the retina.</a> Biochem Soc Trans. 39 (5), 1207-1210 (2011).</li>
<li>Hollo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+regulatory+mechanism+of+human+myosin-7a%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Molecular regulatory mechanism of human myosin-7a.</a> J Biol Chem. 299 (10), 105243(2023).</li>
<li>Choi, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+small+EF-hand+protein+CALML4+functions+as+a+critical+myosin+light+chain+within+the+intermicrovillar+adhesion+complex%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">The small EF-hand protein CALML4 functions as a critical myosin light chain within the intermicrovillar adhesion complex.</a> J Biol Chem. 295 (28), 9281-9296 (2020).</li>
<li>Ebrahim, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stereocilia-staircase+spacing+is+influenced+by+myosin+III+motors+and+their+cargos+espin-1+and+espin-like%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Stereocilia-staircase spacing is influenced by myosin III motors and their cargos espin-1 and espin-like.</a> Nat Commun. 7, 10833(2016).</li>
<li>Whatley, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Usher+syndrome%3A+Genetics+and+molecular+links+of+hearing+loss+and+directions+for+therapy%5BTitle%5D)+AND+%22Front+Genet%22%5BJournal%5D)">Usher syndrome: Genetics and molecular links of hearing loss and directions for therapy.</a> Front Genet. 11, 565216(2020).</li>
<li>Ahmed, Z. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((USH1H%2C+a+novel+locus+for+type+I+Usher+syndrome%2C+maps+to+chromosome+15q22-23%5BTitle%5D)+AND+%22Clin+Genet%22%5BJournal%5D)">USH1H, a novel locus for type I Usher syndrome, maps to chromosome 15q22-23.</a> Clin Genet. 75 (1), 86-91 (2009).</li>
<li>Liu, X., Ondek, B., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutant+myosin+VIIa+causes+defective+melanosome+distribution+in+the+RPE+of+shaker-1+mice%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Mutant myosin VIIa causes defective melanosome distribution in the RPE of shaker-1 mice.</a> Nat Genet. 19 (2), 117-118 (1998).</li>
<li>Gibbs, D., Kitamoto, J., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abnormal+phagocytosis+by+retinal+pigmented+epithelium+that+lacks+myosin+VIIa%2C+the+Usher+syndrome+1B+protein%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein.</a> Proc Natl Acad Sci U S A. 100 (11), 6481-6486 (2003).</li>
<li>Houdusse, A., Titus, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+many+roles+of+myosins+in+filopodia%2C+microvilli+and+stereocilia%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">The many roles of myosins in filopodia, microvilli and stereocilia.</a> Curr Biol. 31 (10), R586-R602 (2021).</li>
<li>Schwander, M., Kachar, B., Muller, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+series%3A+The+cell+biology+of+hearing%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Review series: The cell biology of hearing.</a> J Cell Biol. 190 (1), 9-20 (2010).</li>
<li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin-VIIa+is+expressed+in+multiple+isoforms+and+essential+for+tensioning+the+hair+cell+mechanotransduction+complex%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Myosin-VIIa is expressed in multiple isoforms and essential for tensioning the hair cell mechanotransduction complex.</a> Nat Commun. 11 (1), 2066(2020).</li>
<li>Fitzgerald, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+complex+expression+by+using+multigene+baculoviral+vectors%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Protein complex expression by using multigene baculoviral vectors.</a> Nat Methods. 3 (12), 1021-1032 (2006).</li>
<li>Sari, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+MultiBac+baculovirus%2Finsect+cell+expression+vector+system+for+producing+complex+protein+biologics%5BTitle%5D)+AND+%22Adv+Exp+Med+Biol%22%5BJournal%5D)">The MultiBac baculovirus/insect cell expression vector system for producing complex protein biologics.</a> Adv Exp Med Biol. 896, 199-215 (2016).</li>
<li>Courtney, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptotagmin-7+outperforms+synaptotagmin-1+to+promote+the+formation+of+large%2C+stable+fusion+pores+via+robust+membrane+penetration%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration.</a> Nat Commun. 14 (1), 7761(2023).</li>
<li>Courtney, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+complexin+C-terminal+amphipathic+helix+stabilizes+the+fusion+pore+open+state+by+sculpting+membranes%5BTitle%5D)+AND+%22Nat+Struct+Mol+Biol%22%5BJournal%5D)">The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.</a> Nat Struct Mol Biol. 29 (2), 97-107 (2022).</li>
<li>Courtney, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptotagmin+1+oligomerization+via+the+juxtamembrane+linker+regulates+spontaneous+and+evoked+neurotransmitter+release%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Synaptotagmin 1 oligomerization via the juxtamembrane linker regulates spontaneous and evoked neurotransmitter release.</a> Proc Natl Acad Sci U S A. 118 (48), e2113859118(2021).</li>
<li>Yu, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+7+and+its+adaptors+link+cadherins+to+actin%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Myosin 7 and its adaptors link cadherins to actin.</a> Nat Commun. 8, 15864(2017).</li>
<li>Kron, S. J., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+actin+filaments+move+on+myosin+fixed+to+a+glass+surface%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Fluorescent actin filaments move on myosin fixed to a glass surface.</a> Proc Natl Acad Sci U S A. 83 (17), 6272-6276 (1986).</li>
<li>Sellers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+motility+assay+to+study+translocation+of+actin+by+myosin%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">In vitro motility assay to study translocation of actin by myosin.</a> Curr Protoc Cell Biol. Chapter 13, 13.2(2001).</li>
<li>Tripathi, A., Bond, C., Sellers, J. R., Billington, N., Takagi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin-specific+adaptations+of+in+vitro+fluorescence+microscopy-based+motility+assays%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Myosin-specific adaptations of in vitro fluorescence microscopy-based motility assays.</a> J Vis Exp. (168), e62180(2021).</li>
<li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+FERM+domain+autoregulates+Drosophila+myosin+7a+activity%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">A FERM domain autoregulates Drosophila myosin 7a activity.</a> Proc Natl Acad Sci U S A. 106 (11), 4189-4194 (2009).</li>
<li>Jorio, H., Tran, R., Kamen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stability+of+serum-free+and+purified+baculovirus+stocks+under+various+storage+conditions%5BTitle%5D)+AND+%22Biotechnol+Prog%22%5BJournal%5D)">Stability of serum-free and purified baculovirus stocks under various storage conditions.</a> Biotechnol Prog. 22 (1), 319-325 (2006).</li>
<li>JoVE Science Education Database. Separating protein with SDS-PAGE. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22Basic+methods+in+cellular+and+molecular+biology%22">Basic methods in cellular and molecular biology</a>. , JoVE, Cambridge, MA. (2023).</li>
<li>Mikolajczyk, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+ubiquitin-related+modifier+%28SUMO%29-specific+proteases%3A+profiling+the+specificities+and+activities+of+human+SENPs%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Small ubiquitin-related modifier (SUMO)-specific proteases: profiling the specificities and activities of human SENPs.</a> J Biol Chem. 282 (36), 26217-26224 (2007).</li>
<li>Pardee, J. D., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+muscle+actin%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Purification of muscle actin.</a> Methods Enzymol. 85 Pt B, 164-181 (1982).</li>
<li>Aksel, T., Choe Yu, E., Sutton, S., Ruppel, K. M., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ensemble+force+changes+that+result+from+human+cardiac+myosin+mutations+and+a+small-molecule+effector%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Ensemble force changes that result from human cardiac myosin mutations and a small-molecule effector.</a> Cell Rep. 11 (6), 910-920 (2015).</li>
<li>Bowser, R. M., Farman, G. P., Gregorio, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Philament%3A+A+filament+tracking+program+to+quickly+and+accurately+analyze+in+vitro+motility+assays%5BTitle%5D)+AND+%22Biophys+Rep%22%5BJournal%5D)">Philament: A filament tracking program to quickly and accurately analyze in vitro motility assays.</a> Biophys Rep. 4 (1), 100147(2024).</li>
<li>Bird, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chaperone-enhanced+purification+of+unconventional+myosin+15%2C+a+molecular+motor+specialized+for+stereocilia+protein+trafficking%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.</a> Proc Natl Acad Sci U S A. 111 (34), 12390-12395 (2014).</li>
<li>Billington, N., Wang, A., Mao, J., Adelstein, R. S., Sellers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+three+full-length+human+nonmuscle+myosin+II+paralogs%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">Characterization of three full-length human nonmuscle myosin II paralogs.</a> J Biol Chem. 288 (46), 33398-33410 (2013).</li>
<li>Billington, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+18A+coassembles+with+nonmuscle+myosin+2+to+form+mixed+bipolar+filaments%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.</a> Curr Biol. 25 (7), 942-948 (2015).</li>
<li>Lu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Competition+between+kinesin-1+and+myosin-V+defines+Drosophila+posterior+determination%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Competition between kinesin-1 and myosin-V defines Drosophila posterior determination.</a> Elife. 9, 54216(2020).</li>
<li>Liu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+binding+protein+regulates+myosin-7a+dimerization+and+actin+bundle+assembly%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">A binding protein regulates myosin-7a dimerization and actin bundle assembly.</a> Nat Commun. 12 (1), 563(2021).</li>
<li>Heissler, S. M., Sellers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+light+chains%3A+Teaching+old+dogs+new+tricks%5BTitle%5D)+AND+%22Bioarchitecture%22%5BJournal%5D)">Myosin light chains: Teaching old dogs new tricks.</a> Bioarchitecture. 4 (6), 169-188 (2014).</li>
<li>Belyaev, A. S., Hails, R. S., Roy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-level+expression+of+five+foreign+genes+by+a+single+recombinant+baculovirus%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">High-level expression of five foreign genes by a single recombinant baculovirus.</a> Gene. 156 (2), 229-233 (1995).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ca%282%2B%29-induced+rigidity+change+of+the+myosin+VIIa+IQ+motif-single+alpha+helix+lever+arm+extension%5BTitle%5D)+AND+%22Structure%22%5BJournal%5D)">Ca(2+)-induced rigidity change of the myosin VIIa IQ motif-single alpha helix lever arm extension.</a> Structure. 25 (4), 579-591.e4 (2017).</li>
<li>Persson, M., Bengtsson, E., ten Siethoff, L., Mansson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonlinear+cross-bridge+elasticity+and+post-power-stroke+events+in+fast+skeletal+muscle+actomyosin%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Nonlinear cross-bridge elasticity and post-power-stroke events in fast skeletal muscle actomyosin.</a> Biophys J. 105 (8), 1871-1881 (2013).</li>
</ol>

The authors declare no conflict of interest.

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 types of light chains, all of which are essential for the protein&#39;s structural and functional integrity14. This is in contrast to its invertebrate homolog and most other myosins, which typically bind to only one or two light chain types44,45. This means that to synthesize the human myosin-7a holoenzyme, at least four different genes must be expressed simultaneously in each Sf9 cell. In this case, the MultiBac system offers significant benefits over co-infection with multiple baculoviruses because it ensures reproducible ratios of the myosin-7a complex subunits in each infected cell. In fact, Belyaev et al. have statistically demonstrated this: as the number of virus types increases, the likelihood of cells receiving an equal virus ratio decreases dramatically46. For example, with two virus types, only 12.78% of cells achieve an equal ratio, while this percentage drops to 0.29% when four virus types are used, assuming each virus type is distributed between cells independently. This variability can be problematic when the subunit proteins need to assemble at a consistent ratio to produce the maximum yield of the complex. While this paper focuses on mammalian myosin-7a, it is increasingly evident that the MultiBac system offers a more optimized approach for producing multimeric complexes than the co-infection method, particularly for proteins with more subunits and produced in low yields.
Several factors are critical for achieving high-yield production of myosin-7a protein using this method. First, it is crucial to determine the optimal timing for harvesting Sf9 cells. This allows maximum protein production while minimizing the detrimental effects of cell lysis and death caused by the virus. MultiBac-based protein complex production often exhibits a delayed peak in expression compared to monocistronic baculovirus systems or when expressing smaller proteins. We found that the best time to harvest cells infected with the myosin-7a MultiBac virus is between 60 - 65 h post-infection. Continuous monitoring of protein expression levels throughout the process is strongly recommended. This can be accomplished using fluorescent microscopy if a fluorescent protein tag is fused or through SDS-PAGE analysis otherwise. Additionally, it is important to monitor cell viability concurrently to identify the optimal timing for achieving the highest protein yield with minimal cell death.
Myosin-7a is a large monomeric myosin susceptible to protein degradation14. To prevent degradation during the purification process, it is important to ensure that all buffers are pre-chilled and all procedures are conducted at 4 &#730;C. In addition, minimizing the exposure of myosin-7a to proteases is critical. Besides utilizing protease inhibitor cocktails, we recommend keeping the incubation of the cell lysate with FLAG-resin to just 1 h. Extending this duration has not been shown to significantly improve resin binding or increase total protein yield and may pose a higher risk of protein degradation.
A distinctive feature of mammalian myosin-7a, compared to isoforms from lower species44, is its combination of unique light chain components. These light chains play important regulatory roles in the function of myosin-7a. For instance, calmodulin dynamically interacts with the myosin-heavy chain in a Ca2+-dependent manner47, modulating its motility and mechanical output, a mechanism that seems specifically adapted to the mammalian auditory hair cells14. While the exact binding affinities of calmodulin to individual IQ motifs have yet to be determined in the context of the whole molecule, we have observed that some calmodulin gradually dissociates as the excess light chains in the cell lysate are removed during purification. This may alter the mechanical properties of myosin-7a. To mitigate this, we employ spin columns combined with gentle centrifugation for the elution step. This allows proteins to be eluted in a small volume and at a high concentration, which can obviate the need for concentration steps. This practice shortens the total protein purification process and minimizes the risk of light chain dissociation. In actin gliding assays, we include excess light chain proteins in the final buffer to ensure that the native light chains remain bound with the myosin-7a heavy chain.
The requirement for excess light chains represents one of several differences between the actin gliding assay with myosin-7a and that of other well-studied myosins such as myosin-2. Myosin-2 assays are typically performed at low ionic strength (e.g., 50 mM). As ionic strength is raised towards physiological (150 mM), actin filaments tend to detach, and motility slows. In the case of myosin-7a, motility is stalled at low ionic strength, and gliding is only observed at greater than or equal to 150 mM. Due to the autoinhibition of full-length myosin-7a, it is applied to the coverslip in a high ionic strength solution in a manner akin to how myosin filaments are dissolved before application to allow proteins to bind in an orientation and conformation that allows subsequent gliding.
The low velocity observed in actin gliding assays with myosin-7a introduces some challenges in acquiring and analyzing motility data. Indeed, the translocation is several orders of magnitude slower compared to skeletal muscle myosin, which was used when this assay was originally developed30. This low velocity can lead to difficulty in accurate measurement and an overestimation of velocity that scales with frame rate48. The localization precision of any tracking method is finite, and even a static object has an apparent shift in position between successive images. As the sampling rate increases, this leads to an artificially high value for velocity. This effect is true for any type of motility assay, but the relative effect is very small for assays where a large movement of several pixels occurs between frames. By taking data points at very long intervals (every 60 s, 90 s, etc.), it can be verified that the measured velocity is accurate since the value should be unaffected by the sampling rate. Since the recording interval for myosin-7a is so long, the delay can be taken advantage of to record from several positions during the same acquisition. This effectively allows for the recording of several movies at one time. A disadvantage of this method is that mechanical imperfections with the stage will lead to additional drift due to the switching of positions. This can be accounted for using image stabilization as described above.
In the original Python2 version of the FAST software (github.com/turalaksel/FASTrack), each output data point represents a velocity for a filament within an N-frame window (5 frames by default). A modified Python3 version of the software (github.com/NeilBillington/FASTrack3) includes an additional output in which data points are on a filament-by-filament basis. The default plots produced by the software are based on the N window-type dataset. Both output types are equally valid, and although the original output will produce many more data points per movie, we typically use the filament-based data since this is more directly comparable to existing methods for quantifying filament gliding and yields more intuitive information about the numbers of filaments with specific velocities in a particular movie. Note that even in this filament-type analysis, the number of data points will not correspond exactly to the true number of filaments since tracking stops when filaments cross paths, and new tracks are then detected as they emerge after crossing.
Limitations of the methods described in this paper are that mammalian protein is being expressed in insect cells. Although many such proteins, including this one, have been successfully expressed in this way, there are other expression systems that may more closely mimic the native environment of the protein. Mammalian expression systems could introduce important post-translational modifications to the protein which are absent in the insect system. The same is true to an even greater extent for expression in a bacterial system, as used here for producing myosin light chains. Nevertheless, we believe the relative simplicity and high yield in these cases outweigh the potential limitations. The in vitro motility assay that is used to characterize the protein is limited in how much information it can yield about the protein. For example, many aspects of myosin regulation are masked or circumvented by the assay and thus cannot be investigated using this technique. Many other assay types, such as single-molecule motility, optical trapping, and biochemical and biophysical techniques, exist to investigate the properties of myosin in vitro, but the filament gliding assay is chosen here as a method to measure myosin activity because it requires less protein than many biochemical assays, is simple to perform and where successful motility can be demonstrated, tells us that the protein is both biochemically and mechanically active.
The workflow described here represents a series of methods for producing high-quality myosin-7a protein and characterizing its mechanical properties. Although these methods are specifically tailored to this myosin class, the expression and purification procedures are more generally useful as a guideline for how to produce this type of labile protein, consisting of several distinct polypeptides and with a tendency for dissociation and degradation. In addition, the characterization methods are useful for all types of motor proteins, and in particular, the considerations of data acquisition and analysis parameters are intended to be useful for anyone measuring the translocation rate of low-velocity molecular motors.

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><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 (&gt;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;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;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&#039;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

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.

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.
Video 1 and Figure 5 demonstrate a characteristic actin filament gliding assay with mammalian myosin-7a. An immediate feature revealed by this assay is the slow motility of myosin-7a. The video is played back at 500 times normal speed to better visualize the movement of actin filaments driven by this myosin. This assay can be modified to further study how external factors such as temperature, ionic strength, and solution viscosity influence the activity of myosin-7a. Additionally, myosin-7a binding proteins can be introduced into the flow cell to investigate their effects on actomyosin interactions and motility.
<img alt="Baculovirus expression system diagram; plasmid construction, ligation, virus amplification process." class="xfigimg" src="/files/ftp_upload/67135/67135fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: Workflow for MultiBac-system-based myosin-7a holoenzyme production. (A) Insert the cDNAs encoding the myosin-7a heavy chain, RLC, CALML4, and calmodulin into the multiple cloning sites (MCS) of the pACEBac1 vector. (B) Construct the multigene expression cassette for encoding the myosin-7a complex by iteratively ligating the pACEBac1 vectors that contain the cDNAs of the myosin-7a heavy chain, RLC, CALML4, and calmodulin. (C) Integrate the myosin-7a multigene cassette into the baculovirus genome through the transposition of the mini-Tn7 element from the pACEBac1 vector to the mini-attTn7 target site in the genome. (D) Produce baculoviruses expressing the myosin-7a complex by transfecting Sf9 cells with the recombinant bacmid containing the myosin-7a multigene cassette. (E) The baculovirus can be amplified by inoculating the P0 virus into an Sf9 cell suspension culture and collecting the supernatant. The resulting product, the P1 virus, can be used for large-scale protein expression. (F) Workflow for the FLAG-affinity column-based purification of the myosin-7a complex. <a href="https://www.jove.com/files/ftp_upload/67135/67135fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Fluorescence microscopy images showing cell culture growth over time; scale bars at 100 μm." class="xfigimg" src="/files/ftp_upload/67135/67135fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Representative fluorescent images of Sf9 cells transfected with bacmid expressing myosin-7a with a C-terminal GFP tag. The images show the normal progression of baculovirus infection: cells begin to express myosin-7a around day 4 post-transfection, with nearly all cells becoming infected within one week. <a href="https://www.jove.com/files/ftp_upload/67135/67135fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Polyacrylamide gel electrophoresis results of protein separation showing molecular weight markers." class="xfigimg" src="/files/ftp_upload/67135/67135fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: SDS-gels of purified myosin-7a complex and light chain proteins. (A) Purified full-length human myosin-7a holoenzyme using the MultiBac system. The heavy chain (HC) and light chains are indicated. (B) Purified RLC and CALML4 using the His6-SUMO system. Calmodulin (CaM) is purchased (see Table of Materials) and purified from the Bovine brain. <a href="https://www.jove.com/files/ftp_upload/67135/67135fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Static equilibrium experiment diagram with nitrocellulose coverslip setup for microscopy." class="xfigimg" src="/files/ftp_upload/67135/67135fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Workflow for flow chamber assembly and actin gliding assays. A nitrocellulose-treated coverslip is attached to a microscope slide using two pieces of double-sided tape. This creates a flow chamber with a volume of approximately 10 µL. Proteins are added sequentially to the chamber, while excess solutions are wicked through the channel using tissue paper. <a href="https://www.jove.com/files/ftp_upload/67135/67135fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Actin filament analysis; velocity histogram and data plots; velocity vs. filament length; microscopy." class="xfigimg" src="/files/ftp_upload/67135/67135fig05.jpg" data-altupdatedat="1788304762000" data-alt="Figure 5"> 
Figure 5: Representative results of gliding actin filament assays and the tracking analysis. (A) Example frame from a timelapse recording showing actin filament movement driven by surface-decorated myosin-7a motors. (B) Filament tracking image output from the FAST program for the same field-of-view as shown in (A). (C) Representative histogram of the actin gliding velocity generated by mammalian myosin-7a: 4.2 ± 1.4 nm/s (mean ± standard deviation; the number of tracks = 550). (D) Example of autogenerated output from the FAST program showing the calculated filament length and velocities. %STUCK shows the percentage of filaments that are deemed to be stuck based on the user supplied minimum velocity cutoff. MVEL represents the mean velocity of all non-stuck filaments. <a href="https://www.jove.com/files/ftp_upload/67135/67135fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Actin filament gliding assay performed with myosin-7a. Purified full-length human myosin-7a is attached to the surface. This movie was acquired at 1 frame every 30 s with 200 ms exposure. <a href="https://www.jove.com/files/ftp_upload/67135/JOVE_Video1_New.mp4" target="_blank">Please click here to download this Video.</a>

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

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JoVE Journal

Biochemistry

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

The MultiBac Protein Complex Production Platform at the EMBL

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Laboratory Scale Production and Purification of a Therapeutic Antibody

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Expression, Purification, and Antimicrobial Activity of S100A12

We present approaches for the biophysical and structural characterization of glycoproteins with the immunoglobulin fold by biolayer interferometry, isothermal titration calorimetry, and X-ray crystallography.

Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques

This article describes specific methods to obtain biochemical quantities of detergent-solubilized TRPV1 for spectroscopic analysis. The combined protocols provide biochemical and biophysical tools that can be adapted to facilitate structural and functional studies for mammalian ion channels in a membrane-controlled environment.

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

Expression and Purification of Mammalian Bestrophin Ion Channels

Learn to purify human Myosin-7a using the MultiBac Baculovirus system and study its motility with in vitro assays. This method enables investigation of hearing mechanisms and deafness-related molecular defects.

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

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