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

Summary

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.

Abstract

Rhipicephalus microplus – the cattle tick – 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.

Introduction

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 piroplasmosis^1,2,3,4. 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 chem....

Protocol

1. Dissection of the Gut Epithelium from R. microplus

1. Collect semi-engorged ticks from cattle on the day of experiment. Dissect ticks within 24 h after removal from the host.
2. 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.
3. Pour 100 mL of phosphate buffered saline (PBS) into the petri dish, or until the tick is completely submerged.
4. Utilizing a size 11 scalpel, cut from the top of the eyes to the bottom festoons, on both sides of the tick.
5. Using sterile ....

Results

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.......

Discussion

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 acaricides^1,4. 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.......

Materials

List of materials used in this article

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