<meta charset="utf8"></head><body>重组人肌球蛋白 7a 生产和功能表征方案

<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

<meta charset="utf8"></head><body>作者聚焦：揭示肌球蛋白 7a 和 Usher 蛋白在听力和人类疾病中的作用

基于 MultiBac 系统的人肌球蛋白 7a 纯化和生物物理表征

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

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

Research

JoVE Journal

Biochemistry

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

338 次观看.  West Virginia University. 我们试图回答为什么肌球蛋白马达的突变会导致人类疾病，以及这些蛋白质在听觉过程中起什么作用。我们的研究重点是了解肌球蛋白 7a 的分子机制，以及它如何与 Usher 蛋白一起组装和维持听力所需的细胞机制。了解 Myosin-7a 的结构和功能机制需要高质量的完整蛋白，这一直是该领域的一个长期挑战。最近，我们取得了重大进展，成功纯化了?...

视频: 作者聚焦：揭示肌球蛋白 7a 和 Usher 蛋白在听力和人类疾病中的作用

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

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

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

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.

热稳定，表达，纯化，和人类血清素转运蛋白结晶绑定到<em&gt;取值</em&gt; -citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

科研

生物化学

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

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.

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

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

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

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

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.

重组蛋白表达，结晶和生物物理研究<em&gt;芽孢杆菌</em&gt; - 保守的核苷酸焦磷酸化酶，BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

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

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of these glycoproteins possess immunoglobulin (Ig) folds and are central to immune recognition and regulation. Here, we present a platform for the design, expression and biophysical characterization of the extracellular domain of human B cell receptor CD22. We propose that these approaches are broadly applicable to the characterization of mammalian glycoprotein ectodomains containing Ig domains. Two suspension human embryonic kidney (HEK) cell lines, HEK293F and HEK293S, are used to express glycoproteins harbouring complex and high-mannose glycans, respectively. These recombinant glycoproteins with different glycoforms allow investigating the effect of glycan size and composition on ligand binding. We discuss protocols for studying the kinetics and thermodynamics of glycoprotein binding to biologically relevant ligands and therapeutic antibody candidates. Recombinant glycoproteins produced in HEK293S cells are amenable to crystallization due to glycan homogeneity, reduced flexibility and susceptibility to endoglycosidase H treatment. We present methods for soaking glycoprotein crystals with heavy atoms and small molecules for phase determination and analysis of ligand binding, respectively. The experimental protocols discussed here hold promise for the characterization of mammalian glycoproteins to give insight into their function and investigate the mechanism of action of therapeutics.

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.

用 X 射线晶体学和生物物理技术表征免疫球蛋白折叠的糖蛋白

Glycoproteins on the surface of cells play critical roles in cellular function, including signalling, adhesion and transport. On leukocytes, several of ...

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

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.

光谱分析 TRPV1 的纯化与重构

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as membrane proteins. The experimental procedures for the co-translational insertion and folding of membrane proteins into preformed and defined membranes supplied as nanodiscs are demonstrated. The protocol is completely detergent-free and can generate milligrams of purified samples within one day. The resulting membrane protein/nanodisc samples can be used for a variety of functional studies and structural applications such as crystallization, nuclear magnetic resonance, or electron microscopy. The preparation of basic key components such as cell-free lysates, nanodiscs with designed membranes, critical stock solutions as well as the assembly of two-compartment cell-free expression reactions is described. Since folding requirements of membrane proteins can be highly diverse, a major focus of this protocol is the modulation of parameters and reaction steps important for sample quality such as critical basic reaction compounds, membrane composition of nanodiscs, redox and chaperone environment, or DNA template design. The whole process is demonstrated with the synthesis of proteorhodopsin and a G-protein coupled receptor.

Co-translational insertion into pre-formed nanodiscs makes it possible to study cell-free synthesized membrane proteins in defined lipid environments without contact with detergents. This protocol describes the preparation of essential system components and the critical parameters for improving expression efficiency and sample quality.

膜蛋白共翻译插入预成型纳米盘

Cell-free expression systems allow the tailored design of reaction environments to support the functional folding of even complex proteins such as ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

神经节苷脂提取、纯化和分析

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Bacterial Expression and Purification of Human Matrix Metalloproteinase-3 using Affinity Chromatography

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, as well as interactions with several growth factors and cytokines. Overexpression of specific MMPs is responsible in several diseases such as cancer, neurodegenerative diseases, and cardiovascular disease. MMPs have been the center of attention recently as targets to develop therapeutics that can treat diseases correlated to MMP overexpression.
To study the MMP mechanism in solution, more facile and robust recombinant protein expression and purification methods are needed for the production of active, soluble MMPs. However, the catalytic domain of most MMPs cannot be expressed in Escherichia coli (E. coli) in soluble form due to lack of posttranslational machinery, whereas mammalian expression systems are usually costly and have lower yields. MMP inclusion bodies must undergo the tedious and laborious process of extensive purification and refolding, significantly reducing the yield of MMPs in native conformation. This paper presents a protocol using Rosetta2(DE3)pLysS (hereafter referred to as R2DP) cells to produce matrix metalloproteinase-3 catalytic domain (MMP-3cd), which contains an N-terminal His-tag followed by pro-domain (Hisx6-pro-MMP-3cd) for use in affinity purification. R2DP cells enhance the expression of eukaryotic proteins through a chloramphenicol-resistant plasmid containing codons normally rare in bacterial expression systems. Compared to the traditional cell line of choice for recombinant protein expression, BL21(DE3), purification using this new strain improved the yield of purified Hisx6-pro-MMP-3cd. Upon activation and desalting, the pro domain is cleaved along with the N-terminal His-tag, providing active MMP-3cd for immediate use in countless in vitro applications. This method does not require expensive equipment or complex fusion proteins and describes rapid production of recombinant human MMPs in bacteria.

His-tag purification, dialysis, and activation are employed to increase yields of soluble, active matrix metalloproteinase-3 catalytic domain protein expression in bacteria. Protein fractions are analyzed via SDS-PAGE gels.

亲和色谱法表达和纯化人基质金属蛋白酶-3

Matrix metalloproteinases (MMPs) belong to the family of metzincin proteases with central roles in extracellular matrix (ECM) degradation and remodeling, ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

在膜上重构隔膜组件以研究生物物理性质和功能

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

表达和纯化溶质载体转运蛋白的策略

High-Throughput Expression and Purification of Human Solute Carriers for Structural and Biochemical Studies

Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using&#160;codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.

作者聚焦：使用密码子优化基因表达和纯化人溶质载体转运蛋白 

Structural and biochemical studies of human membrane transporters require milligram quantities of stable, intact, and homogeneous protein. Here we describe scalable methods to screen, express, and purify human solute carrier transporters using codon-optimized genes.