Name: purification of biotinylated cell surface proteins from rhipicephalus microplus epithelial gut cells
Uploaded: 2017-07-23T14:00:00.000+00:00
Duration: 12 min 3 s
Description: Watch this Scientific Journal Video about Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells at JoVE.com

The authors wish to thank the Biosecurity Tick Colony (Queensland Department of Agriculture &amp; Fisheries, Australia) for the provision of Rhipicephalus microplus ticks utilized for this study, and Lucas Karbanowicz for assistance with video filming.

1. Dissection of the Gut Epithelium from R. microplus

<ol>
	<li>Collect semi-engorged ticks from cattle on the day of experiment. Dissect ticks within 24 h after removal from the host.</li>
	<li>Adhere a strip of duct tape to the bottom of the 92 mm x 16 mm Petri dish. Add a drop of super glue to the tape. Place the tick, ventral side down on the super glue, allow to dry for 2 min.</li>
	<li>Pour 100 mL of phosphate buffered saline (PBS) into the petri dish, or until the tick is completely submerged.</li>
	<li>Utilizing a size 11 scalpel, cut from the top of the eyes to the bottom festoons, on both sides of the tick.</li>
	<li>Using sterile forceps, completely remove the scutum and alloscutum, to expose the internal organs.</li>
	<li>Remove the fine white thread-like organs (trachea) and other membranes to prevent contamination.</li>
	<li>Remove the gut using forceps, by pinching the upper region and pulling from the carcass. Remove any remaining gut tissues, ensuring that no other tissues have been dissected.</li>
	<li>Store gut in ice-cold Hank&#39;s Balanced Salt Solution (HBSS) without calcium chloride and magnesium sulfate with proteinase-inhibitor cocktail (PIC). Snap freeze the guts in dry ice and store at -20 &#176;C. 
	Note: To assist with gut dissection and details of the tick internal organs refer to Chapter 3.1 of D. Sonenshine, &#34;Biology of Ticks&#34;19.</li>
</ol>
2. Epithelial Cell Dissociation
<ol>
	<li>Pour the dissected gut onto a 70 &#181;m cell strainer inside a 50 mL tube.</li>
	<li>Flush the gut tissue with 50 mL ice cold HBSS with PIC until the solution runs clear, and the guts take on a white/clear appearance.</li>
	<li>Re-suspend guts in 30 mL ice cold HBSS with PIC, mix gently and centrifuge at 500 x g at 4 &#176;C for 10 min to pellet the gut. Remove the supernatant and repeat the wash process three times.</li>
	<li>To dislodge epithelial gut cells, re-suspend the gut in 10 mL of cell culture medium Dulbecco&#39;s modified eagle medium (DMEM), 2% fetal calf serum (FCS), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mM Tris(2-carboxyethyl)phosphine (TCEP), PIC and incubate for 60 min at 37 &#176;C under slow rotation using a roller at 6 rpm.</li>
	<li>Filter the suspension through a 250 &#181;m cell strainer, vortex the flow-through and filter through a 70 &#181;m cell strainer collecting the remaining flow-through.</li>
	<li>Centrifuge the suspension at 500 x g at 4 &#176;C for 20 min to pellet the single cells.</li>
</ol>
3. Isolation of Single Epithelial Cells using a density centrifugation gradient
<ol>
	<li>Prepare density centrifugation gradient (e.g., Percoll) by filtering through an AP15 pre-filter paper. Prepare 40% and a 20% Percoll in mqH20 (v/v) and cool at 4 &#176;C for 1 h prior to layering the gradient.</li>
	<li>Using a peristaltic pump set at the lowest speed, layer 3 mL of 40% density centrifugation gradient into a 16 mL ultracentrifuge tube, allowing it to settle on ice for 15 min. Speed of the pump should lead to a &#60;1 mL per min flow rate.</li>
	<li>Tilting the tube to a 45&#176; angle, use the peristaltic pump to layer the 20% density centrifugation gradient on top of the 40% layer. Speed of the pump should lead to &#60;1 mL per min flow rate. Allow the layers to settle on ice for 15 min.</li>
	<li>Use the peristaltic pump at &#60;1 mL per min flow rate to layer 3 mL of DMEM medium containing tick gut cells over the 20-40% density centrifugation gradient.</li>
	<li>Program the centrifuge for maximum acceleration and minimum deceleration. Centrifuge at 600 x g for 10 min. Collect interphases between the DMEM:20% density centrifugation gradient, and the 20%: 40% density centrifugation gradients to isolate epithelial single cells. Store collected interphases at 4 &#176;C for subsequent analyses.</li>
</ol>
4. Assessment of Cell Isolation
<ol>
	<li>Hemacytometer
	<ol>
		<li>Clean the hemacytometer slide with alcohol.</li>
		<li>.Re-suspend the cells by gently pipetting the cells up and down. Pipette 100 &#181;L of the cell suspension and place in a 1.5 mL microcentrifuge tube.</li>
		<li>Add 400 &#181;L of 0.4% Trypan Blue. Gently mix by flicking the tube.</li>
		<li>Pipette 100 &#181;L of the Trypan Blue-treated cell suspension to slowly fill both chambers of the hemocytometer.</li>
		<li>Place the hemocytometer under a light microscope, focusing upon the grid lines of the hemocytometer with a 10X objective.</li>
		<li>Using a hand-held tally counter, count the live unstained cells within a set of 16 squares. Within the same square, count the blue dead cells. Continue counting until four sets of 16 squares are counted.</li>
		<li>Calculate the total cells per mL by using the formula: 
		<img alt="Equation 1" src="/files/ftp_upload/55747/55747eq1.jpg" /></li>
		<li>Calculate the percentage cell viability by using the formula: 
		<img alt="Equation 2" src="/files/ftp_upload/55747/55747eq2.jpg" /></li>
	</ol>
	</li>
	<li>Cell Isolate Visualization
	<ol>
		<li>Dilute 1 &#181;L of the isolated cells in 9 &#181;L of HBSS in a 1.5 mL microcentrifuge tube. Flick the microcentrifuge tube gently to mix.</li>
		<li>Pipette 5 &#181;L of isolated cells in HBSS into the middle of a glass slide. Apply three drops of mounting medium with 4&#39;, 6-diamidino-2-phenylindole (DAPI).</li>
		<li>Incubate the slide at room temperature for 5 min. Carefully place a cover slip over the preparation, avoiding air bubbles.</li>
		<li>Visualize cells stained with DAPI at excitation 360 nm and emission at 460 nm under a fluorescent microscope.</li>
	</ol>
	</li>
</ol>
5. Cell Surface Protein Biotinylation
<ol>
	<li>Biotinylate 100 &#181;L of single cell epithelial cells using Biotin (Type A) Conjugation kit, at a molar ratio of 1:1 surface protein to conjugate, as per manufacturer&#39;s instructions</li>
	<li>For cell lysis, add 100 &#181;L of PBS, 1% Triton X-100, 10% glycerol, 100 &#181;M oxidized glutathione and PIC to the biotinylated cells. Incubate on ice for 1 h with gentle mixing every 10 min.</li>
	<li>Centrifuge the biotinylated cells at 20,000 x g at 4 &#176;C for 20 min to pellet insoluble material. Collect the supernatant containing cytoplasmic, and biotinylated membrane proteins.</li>
	<li>Determine the protein concentration using the Bradford assay.</li>
</ol>
6. Isolation of Biotinylated Surface Proteins
<ol>
	<li>Add 50 &#181;L of Streptavidin Magnetic Beads into a 1.5 mL microcentrifuge tube.</li>
	<li>Place the tube into a magnetic stand, collecting the beads against the side of the tube. Remove and discard the supernatant.</li>
	<li>Add 1000 &#181;L of TBS, 0.1% Tween-20 to the tube. Mix gently and collect the beads with the magnetic stand. Remove and discard the supernatant.</li>
	<li>Combine 40 &#181;g of biotinylated cell surface proteins, diluted to 300 &#181;L in 1x PBS with washed magnetic beads. Incubate for 2 h at room temperature with agitation.</li>
	<li>Collect the beads with a magnetic stand, remove and discard the supernatant.</li>
	<li>Add 300 &#181;L of TBS, 0.1% Tween-20 to the tube, gently mixing to re-suspend the beads. Collect beads, remove and discard the supernatant. Repeat this wash step twice.</li>
	<li>Add 100 &#181;L of the 0.1 M glycine pH 2.0 to the magnetic beads, and incubate at room temperature for 5 min. Collect beads and remove supernatant containing eluted biotinylated surface proteins.</li>
	<li>Visualize isolated surface proteins on a 4-20% Tris-MOPS SDS-PAGE gel.</li>
</ol>
7. Assessment of Biotinylated Surface Protein
<ol>
	<li>Dot Blot
	<ol>
		<li>Cut a 7 cm x 3 cm strip of nitrocellulose membrane.</li>
		<li>Apply 10 &#181;g total tick gut contents (from 1.8 above), and 10 &#181;g of biotinylated surface protein to the membrane. Allow drying for 15 min at room temperature.</li>
		<li>Transfer to a container and submerge in 100 mL of blocking buffer. Incubate at room temperature with agitation for an hour. Discard blocking buffer.</li>
		<li>Wash nitrocellulose membrane in 100 &#181;L PBS, 0.05% Tween-20 for 5 min with agitation. Discard wash buffer and repeat the washes for three times.</li>
		<li>Incubate in 1/5000 Streptavidin- horseradish peroxidase (HRP)in 100 &#181;L PBS, 0.05% Tween-20 for 2 h with agitation at room temperature, and discard any remaining solution.</li>
		<li>Wash nitrocellulose membrane in 100 &#181;L of PBS, 0.05% Tween-20 for 5 min with agitation. Discard wash buffer and repeat the wash three times.</li>
		<li>For detection, dissolve 1 tablet of 4-chloro-1-napthol in 10 mL of ice cold methanol. Add 4 mL of methanol stock to 20 mL of TBS. Add 10 &#181;L of fresh 30% hydrogen peroxide and immediately apply to nitrocellulose membrane.</li>
		<li>Incubate with agitation at room temperature until substrate produces an insoluble blue end product. This may take between 2-15 min.</li>
		<li>Discard detection solution and wash the membrane three times in 100 &#181;L mqH2O.</li>
	</ol>
	</li>
	<li>ELISA Assay
	<ol>
		<li>Dilute 200 ng of each sample with 400 &#181;L of 100 mM Carbonate coating buffer and coat 2 lanes of the first row (A#) of the ELISA plate (flat bottom wells)</li>
		<li>Add 100 &#181;L of 100 mM carbonate coating buffer to all other corresponding wells in rows (B-H).</li>
		<li>Prepare serial dilutions of each sample by pipetting 100 &#181;L from each well in row A, and transferring into row B. Mix gently by pipetting and avoid producing bubbles. Repeat the dilutions down each row, discarding the final 100 &#181;L from each well in row H.</li>
		<li>Cover the plate with parafilm and incubate at 4 &#176;C overnight.</li>
		<li>Wash the plate with 200 &#181;L PBS, 0.05% Tween-20 per well three times.</li>
		<li>Add 200 &#181;L of blocking buffer to the coated wells, cover with Parafilm and incubate at 4 &#176;C overnight.</li>
		<li>Dilute 1/15,000 Streptavidin-HRP in blocking buffer and add 100 &#181;L to each well of the plate. Cover with parafilm and incubate at 4 &#176;C overnight.</li>
		<li>Wash the plate in 200 &#181;L PBS, 0.05% Tween-20 per well five times.</li>
		<li>To detect, add 100 &#181;L of TMB reagent per well. After sufficient color development, usually between 10-15 min, add 100 &#181;L of 1 M phosphoric acid to stop the reaction.</li>
		<li>Read the absorbance of each well at &#955; = 450nm.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Cattle tick infestations constitute a major problem for the cattle industry in tropical and subtropical regions of the world, with the most common method of control reliant upon the use of acaricides1,4. Bm86 was previously identified within the tick gut epithelial surface as a protective antigen against R. microplus infestation10, with limited success as a vaccine strategy due to Bm86 geographic sequence variation and the require...

Rhipicephalus microplus, the cattle tick, is the most significant ectoparasite in terms of economic impact on the cattle industry of tropical and sub-tropical regions as it vectors bovine tick fever (babesiosis), anaplasmosis and equine piroplasmosis1,2,3,4. Efforts have been dedicated to cattle tick control, to diminish the deleterious effect, however conventional methods such as the use of chemical acaricides have implicit drawbacks, such as the presence of chemical residues in milk and meat, and the increase in prevalence of chemically resistant ticks5,6,7. Consequently, the development of alternative methods of tick control have been studied, such as the use of natural resistance cattle, biological control (biopesticides) and vaccines4,5,6,7,8,9.
In the pursuit of proteins capable of being utilized as vaccine candidates, current research is focused upon the tick gut. The midgut wall is built from a single layer of epithelial cells resting on a thin basal lamina, with the outside of the basal lamina forming a network of muscle. Light and electron microscope observations indicate that the midgut consists of three types of cells: reserve (undifferentiated), secretory, and digestive. The number of cell types varies considerably depending upon the physiological phase. Secretory and digestive cells both originate from reserve cells18,19,20.
The construction of cDNA libraries to examine the composition of the tick gut has led to the identification of antigenic proteins, such as Bm86, as potential vaccine candidates2,3,4. The glycoprotein Bm86 is localized at the surface of tick gut cells and induces a protective immune response against the cattle tick (R. microplus) in vaccinated cattle. Anti-Bm86 IgGs produced by the immunized host are ingested by the tick, recognize this antigen on the surface of tick gut cells, and subsequently disturb tick gut tissue function and integrity. Vaccines based upon Bm86 antigens have shown effective control of R. microplus and Rhipicephalus annulatus, by reducing the number, weight and reproductive capacity of engorging females, resulting in a reduced larval infestation in subsequent tick generations4. However, Bm86 based vaccines are not effective against all tick stages and have demonstrated unsatisfactory efficacy against some geographical strains of R. microplus, consequently the beef and dairy industries have poorly adopted these vaccines2,4.
The ability to isolate epithelial cells from the tick gut is a significant innovation which would enable the progression of research to determine protein membrane composition including morphology and physiology under different environmental conditions. The method described here utilizes the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent tris(2-carboxyethyl)phosphine (TCEP) to release the epithelium from its sub-epithelial support tissues10. The epithelium is recovered following mechanical disruption of the tissues by shaking, followed by discontinuous gradient centrifugation in Percoll. This paper describes a feasible and novel technique for the isolation of tick gut epithelial cells. Biotinylated cell surface proteins, isolated from the surface of these epithelial cells can subsequently analyzed in downstream applications such as FACS and/or LC-MS/MS-analysis.

<table><tbody><tr><td>0.4% Trypan Blue</td><td>ThermoFisher Scientific</td><td>15250061</td><td></td></tr><tr><td>1.5 mL microcentrifuge tube</td><td>Eppendorf</td><td>3322</td><td></td></tr><tr><td>100mM Carbonate Buffer</td><td></td><td></td><td>3.03 g Na2CO3, 6.0 g NaHCO3 1000 ml distilled water pH 9.6</td></tr><tr><td>16 mL centrifuge tubes with sealing cap</td><td>Thermo Scientific</td><td>3138-0016</td><td>Cool in ice prior to gradient</td></tr><tr><td>250 &amp;micro;M cell strainer</td><td>Thermo Fisher</td><td>87791</td><td></td></tr><tr><td>3,3&amp;prime;,5,5&amp;prime;-Tetramethylbenzidine (TMB) Liquid Substrate System for ELISA</td><td>Sigma</td><td>T0440</td><td>Stored at 4C</td></tr><tr><td>30% Hydrogen Peroxide</td><td>Labscene</td><td>BSPA5.500</td><td></td></tr><tr><td>4-20% Tris-MOPS Gel</td><td>Gen Script</td><td>M42015</td><td></td></tr><tr><td>4-Chloro-1-naphthol tablet</td><td>Sigma-Aldrich</td><td>C6788</td><td></td></tr><tr><td>50 mL Falcon Tube</td><td>Corning Blue</td><td></td><td>30 x 115mm style. Polyproplyene conical tube.</td></tr><tr><td>70 &amp;micro;M cell strainer</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>AP15 filter paper</td><td>Millipore</td><td>AO1504200</td><td></td></tr><tr><td>Biotin (Type A) Conjugation Kit</td><td>Abcam</td><td>Ab102865</td><td></td></tr><tr><td>Dissection microscope</td><td>Olympus</td><td>SZX7</td><td></td></tr><tr><td>DP Manager&amp;nbsp;</td><td>Olympus</td><td>2.2.1.195</td><td>Cell imagery software</td></tr><tr><td>Duct Tape</td><td>Home Handyman</td><td></td><td>48mm x 25mm Duct Tape</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Gibco</td><td>11995-065</td><td>DMEM - ice cold for protocol</td></tr><tr><td>EDTA</td><td>Amresco</td><td>0105-500G</td><td></td></tr><tr><td>F96 Maxisorp Immuno Plate</td><td>Nunc</td><td>439454</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>12003C</td><td>FCS</td></tr><tr><td>Fluorescence microscope&amp;nbsp;</td><td>&amp;nbsp;Olympus&amp;nbsp;</td><td>BX51</td><td></td></tr><tr><td>Fluoroshield with DAPI</td><td>Sigma-Aldrich</td><td>F6057-20ML</td><td>DAPI</td></tr><tr><td>Forceps</td><td>Dumont</td><td></td><td>#9 Dumont - Switerzland</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td>Glycerol for molecular biology &gt;99%</td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>410225</td><td></td></tr><tr><td>Hand-Held Counter</td><td>Officeworks</td><td>JA0376230</td><td></td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution</td><td>Sigma Life Sciences</td><td>H9394</td><td>HBSS &amp;ndash; ice cold for protocol</td></tr><tr><td>Hemacytometer</td><td>Optik Lakor</td><td>-</td><td>-</td></tr><tr><td>L-Glutathione oxidized</td><td>Sigma-Aldrich</td><td>G4376</td><td></td></tr><tr><td>Magnetic Separation Stand</td><td>Novagen</td><td>-</td><td>4-Tube Magnetic Separation Rack</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>179337</td><td></td></tr><tr><td>Milli-Q Water</td><td>Millipore</td><td>ZRXQ003WW</td><td>Integral Water Purification System for Ultrapure Water</td></tr><tr><td>Nitrocellulose Membrane</td><td>Life Sciences</td><td>66485</td><td>30cm x 3M pure nitrocellulose membrane</td></tr><tr><td>PageRuler Prestained protein Ladder</td><td>Thermo-Fisher</td><td>SM0671</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td>1.16 g Na2HPO4, 0.1 g KCl, 0.1 g K3PO4, 4.0 g NaCl (500 ml distilled water) pH 7.4</td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P1644-500ML</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Masterflex</td><td>7518-10</td><td></td></tr><tr><td>Phosphoric Acid</td><td>Sigma-Aldrich</td><td>P6560</td><td></td></tr><tr><td>Pierce Protein-Free T20 PBS Blocking Buffer</td><td>Thermo-Scientific</td><td>37573</td><td>Stored at 4C. Blocking Buffer</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>P8215-5ML</td><td>PIC &amp;ndash; stored at -20 &lt;b&gt;&amp;deg;&lt;/b&gt;C</td></tr><tr><td>Quick Start Bradford Dye Reagent 1x</td><td>Biorad</td><td>500-0205</td><td>For Bradford Assay</td></tr><tr><td>Quick Start BSA Standards</td><td>Biorad</td><td>500-0207</td><td>BSA standards for Bradford Assay</td></tr><tr><td>Scalpel</td><td>Lab. Co</td><td></td><td>Size 11 Scalpel</td></tr><tr><td>SilverQuest TM Staining Kit</td><td>Invitrogen</td><td>LC6070</td><td></td></tr><tr><td>Simply Blue TM Safe Stain&amp;nbsp;</td><td>Invitrogen</td><td>LC6060</td><td></td></tr><tr><td>Sorvall C6+ Ultracentrifuge</td><td>Thermo Scientific</td><td>46910</td><td></td></tr><tr><td>Streptavidin (HRP)</td><td>Abcam</td><td>AB7403</td><td></td></tr><tr><td>Streptavidin Magnetic Beads</td><td>New England Biolabs</td><td>S1420S</td><td></td></tr><tr><td>Super Glue - Ultra Fast Mini</td><td>UHU</td><td></td><td>UHU Super Glue 1mg. Ultra Fast mini</td></tr><tr><td>Table-top Centrifuge</td><td>Eppendorf</td><td>22331</td><td></td></tr><tr><td>TCEP</td><td>Thermo Fisher</td><td>20490</td><td></td></tr><tr><td>Triton X-100</td><td>Biorad</td><td>161-0407</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P2287-500ML</td><td></td></tr><tr><td>Vortex Mixer</td><td>Ratek</td><td>VM1</td><td></td></tr><tr><td>Water Bath</td><td>Grant</td><td>GD100</td><td></td></tr></tbody></table>

purification of biotinylated cell surface proteins from rhipicephalus microplus epithelial gut cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of several pathogens. Efforts have been dedicated to the cattle tick control to diminish its deleterious effects, with focus on the discovery of vaccine candidates, such as BM86, located on the surface of the tick gut epithelial cells. Current research focuses upon the utilization of cDNA and genomic libraries, to screen for other vaccine candidates. The isolation of tick gut cells constitutes an important advantage in investigating the composition of surface proteins upon the tick gut cells membrane. This paper constitutes a novel and feasible method for the isolation of epithelial cells, from the tick gut contents of semi-engorged R. microplus. This protocol utilizes TCEP and EDTA to release the epithelial cells from the subepithelial support tissues and a discontinuous density centrifugation gradient to separate epithelial cells from other cell types. Cell surface proteins were biotinylated and isolated from the tick gut epithelial cells, using streptavidin-linked magnetic beads allowing for downstream applications in FACS or LC-MS/MS-analysis.

Epithelial cells were isolated from the gut tissues of R. microplus as per the schematic presented in Figure 1. Representative fluorescence microscopy imagery of tick gut epithelial cells prepared using this protocol are shown in Figure 2A and 2B. As the cell isolation is conducted upon semi-engorged R. microplus, cells appear as singular, spher...

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Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Rhipicephalus microplus &#8211; the cattle tick &#8211; is the most significant ectoparasite in terms of economic impact on livestock as a vector of ...

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Biochemistry

9.4K Views.  The University of Queensland. A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.

Video: Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

Cancer Research

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Bioengineering

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Genetics

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Biology

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Immunology and Infection

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at the plasma membrane. Here, we present detailed protocols for 2 methods that facilitate the study of such processes, both of which are based on the principle of the biotinylation of cell-surface proteins. The first is designed to allow for the semi-quantitative determination of the relative levels of a particular protein at the cell-surface. In it, the lysine residues of the plasma membrane proteins of cells are first labeled with a biotin moiety. Once the cells are lysed, these proteins may then be specifically precipitated via the use of agarose-immobilized streptavidin by exploiting the natural affinity of the latter for biotin. The proteins isolated in such a manner may then be analyzed via a standard western blotting approach. The second method provides a means of determining the endocytic rate of a particular cell-surface target over a period of time. Cell-surface proteins are first modified with a biotin derivative containing a cleavable disulfide bond. The cells are then shifted back to normal culture conditions, which causes the endocytic uptake of a proportion of biotinylated proteins. Next, the disulfide bonds of non-internalized biotin groups are reduced using the membrane-impermeable reducing agent glutathione. Via this approach, endocytosed proteins may thus be isolated and quantified with a high degree of specificity.

Two biotinylation-based methods, designed for determining the cell-surface expression and endocytic rate of proteins expressed at the plasma membrane, are presented in this report.

Cell-surface proteins mediate a wide array of functions. In many cases, their activity is regulated by endocytic processes that modulate their levels at ...

Neuroscience

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Cell-surface Biotinylation Assay

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or &#8220;recycled&#8221; back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be chemically cleaved. However, if the surface protein is endocytosed, the biotin derivative will be protected from cleavage. Thus, by analyzing the uncleaved, endocytosed biotin label, scientists can assess the amounts of internalized surface proteins.In this video, we review the concepts behind the biotinylation assay, delving into the chemical structure of the biotin derivative and the mechanism of its cleavage. This is followed by a generalized protocol of the technique, and finally, a description of how researchers are currently using it to study the dynamics of different cell surface proteins.

A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively ...

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Cell Biology

Assessing the Internalization of  Target Surface Proteins Using a Biotin Derivative

Source: Tham, D. K. L., et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/55974">Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation.</a> J. Vis. Exp. (2017).
This video demonstrates a method to assess target protein internalization in the mouse cortical astrocytes using biotinylation, followed by cell lysis, streptavidin-based protein extraction, denaturation, and Western blot analysis to confirm successful internalization.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Determining Internalization ...

Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells

Summary

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Chapters in this video

A Method of Targeted Cell Isolation via Glass Surface Functionalization

Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1^-/- Mice

Determining Cell-surface Expression and Endocytic Rate of Proteins in Primary Astrocyte Cultures Using Biotinylation

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Cell-surface Biotinylation Assay

Assessing the Internalization of Target Surface Proteins Using a Biotin Derivative