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

Summary

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.

Abstract

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.

Introduction

Myosins are molecular motor proteins that interact with actin to drive numerous cellular processes^1,2,3,4. 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 ....

Protocol

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.......

Discussion

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 myosins^40,41,42,43, 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.......

Materials

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