Irga6 Protein Expression and Purification from E. coli

Expression Constructs—The pGW1H-Irga6cTag1 construct was
generated by amplification of the Irga6cTag1 sequence from
pGEX-4T-2-Irga6cTag1 (former pGEX-4T-2-IIGP-m)
(16 (link)) by using Irga6cTag1
forward (5′-cccccccccgtcgaccaccatgggtcagctgttctcttcacctaag-3′) and
reverse (5′-cccccccccgtcgactcagtcacgatgcggccgctcgagtcggcctag-3′)
primers and cloned into pGW1H vector (British Biotech) by SalI digestion.
Mutations were introduced into the coding region of pGW1H-Irga6wt
(15 (link)), pGW1H-Irga6cTag1, and
pGEX-4T2-Irga6wt (16 (link))
according to the QuikChange site-directed mutagenesis kit (Stratagene) using
the following forward and corresponding reverse primers: G2A,
5′-gagtcgaccaccatggctcagctgttctcttca-3′; Δ7-12,
5′-gggtcagctgttctctaataatgatttgccc-3′; Δ7-25,
5′-ccaccatgggtcagctgttctctaaatttaatacggg-3′; Δ20-25,
5′-gaataatgatttgccctccagcaaatttaatacgggaag-3′; F20A,
5′-gagaataatgatttgccctccagcgctactggttattttaag-3′; T21A,
5′-gaataatgatttgccctccagctttgctggttattttaag-3′; G22A,
5′-gccctccagctttactgcttattttaagaaatttaatacggg-3′; Y23A,
5′-gccctccagctttactggtgcttttaagaaatttaatacggg-3′; F24A,
5′-gccctccagctttactggttatgctaagaaatttaatacgggaag-3′; K25A,
5′-gccctccagctttactggttattttgcgaaatttaatacgggaag-3′; K82A,
5′-gggagacgggatcaggggcgtccagcttcatcaataccc-3′; S83N,
5′-ggagacgggatcagggaagaacagcttcatcaataccctg-3′; E106A,
5′-gctaaaactggggtggtggcggtaaccatggaaag-3′.
Cell Culture and Serological Reagents—L929 (CCL-1) and gs3T3
(Invitrogen) mouse fibroblasts were cultured in IMDM or Dulbecco's modified
Eagle's medium (both GIBCO) supplemented with 10% fetal calf serum (Biochrom).
Hybridoma 10D7 and 10E7 cells were grown in IMDM, supplemented with 5% fetal
calf serum. Cells were induced with 200 units/ml IFNγ (Cell Concepts)
for 24 h and transfected using FUGENE6 transfection reagent according to the
manufacturer's protocol (Roche Applied Science). Propagation of T.
gondii strain ME49 was done as described previously
(8 ). gs3T3 cells were infected
for 2 h with T. gondii ME49 strain at a multiplicity of infection of
8 24 h after IFNγ stimulation. The following serological reagents were
used: anti-Irga6 mouse monoclonal antibodies 10D7 and 10E7, anti-Irga6 rabbit
polyclonal serum 165, anti-cTag1 rabbit polyclonal serum 2600
(8 ), donkey-anti-mouse Alexa
546, donkey anti-rabbit Alexa 488 (all from Molecular Probes), goat anti-mouse
κ light chain (Bethyl), goat anti-mouse κ light chain horseradish
peroxidase (Bethyl), goat anti-mouse κ light chain-fluorescein
isothiocyanate (Southern Biotech), 4′,6-diamidino-2-phenylindole (Roche
Applied Science), and donkey anti-rabbit, donkey anti-goat, and goat
anti-mouse horseradish peroxidase (all from Amersham Biosciences).
Antibody Purification and Papain Digestion—10D7 and 10E7
antibodies were purified from corresponding hybridoma supernatants over a
Protein A-Sepharose column (Amersham Biosciences). Antibody was eluted with 50
mm sodium acetate, pH 3.5, 150 mm NaCl and
pH-neutralized to 7.5 with 1 m Tris, pH 11. Buffer was exchanged
five times subsequently by dilution of antibody-containing sample in papain
buffer (75 mm phosphate buffer, pH 7.0, 75 mm NaCl, 2
mm EDTA) and concentrated in a centrifugal concentrator
(Vivaspin20; Sartorius) with a 10 kDa cut-off filter at 2000 ×
g at 4 °C. The concentration of the antibodies was determined by
using formula, concentration of antibody (mg/ml) = 0.8 ×
A₂₈₀. Papain digestion was done according to Ref.
20 (link). The papain-digested
antibodies were further purified on a HiLoad 26/60 Superdex 75 preparation
grade column (Amersham Biosciences) in papain buffer. Samples were incubated
in SDS-PAGE sample buffer under nonreducing conditions and subjected to
SDS-PAGE. Proteins were detected by colloidal Coomassie staining.
Treatment with Aluminum Fluoride—AlCl₃ (Sigma)
was added to 10 ml of IMDM containing no fetal calf serum to a final
concentration of 300 μm and mixed by vigorous shaking.
Subsequently, NaF (Sigma) was added to a final concentration of 10
mm and mixed, and the final solution was applied to confluent L929
cells previously induced with IFNγ or transfected for 24 h. Cells were
incubated in aluminum fluoride complex (AlFx) solution for 30 min at 37 °C
and then washed with cold PBS and collected by scraping. Cell pellets were
lysed in 0.1% Thesit/PBS containing 300 μm AlCl₃ and
10 mm NaF in the presence or absence of 0.5 mm GTP for 1
h at 4 °C.
Immunoprecipitation and
Immunofluorescence—Immunoprecipitation was modified from Ref.
21 . 1 × 10⁶ L929 fibroblasts/sample were induced with IFNγ and/or transfected for 24
h (or left untreated) and harvested by scraping. Cells were lysed in 0.1%
Thesit, 3 mm MgCl₂, PBS, Complete Mini protease
inhibitor mixture without EDTA (Roche Applied Science) for 1 h at 4 °C in
the absence of nucleotide or in the presence of 0.5 mm GDP, GTP,
GTPγS, or 300 μm AlCl₃ and 10 mm NaF
in the presence or absence of 0.5 mm GTP (all from Sigma). Protein
A-Sepharose™ CL-4B beads (Amersham Biosciences) were incubated with 10D7
monoclonal mouse anti-Irga6 antibody or 2600 (anti-cTag1) polyclonal rabbit
serum for 1 h at 4 °C. Bound proteins were eluted by boiling for 10 min in
elution buffer (100 mm Tris/HCl, pH 8.5, 0.5% SDS) with SDS-PAGE
sample buffer (50 mm Tris/HCl, pH 6.1, 1% SDS, 5% glycerol, 0.0025%
bromphenol blue (w/v), 0.7% β-mercaptoethanol). Immunofluorescence was
preformed as previously described
(15 (link)).
FIGURE 1.

10D7 antibody detects Irga6 at the PVM but not at the ER. gs3T3
fibroblasts were induced with IFNγ for 24 h prior to 2-h infection with
T. gondii ME49 strain with a multiplicity of infection of 8. Irga6
protein was labeled with rabbit anti-Irga6 polyclonal serum 165 (red)
and with mouse monoclonal anti-Irga6 antibodies 10E7 (A) or 10D7
(B) (green). PC, phase-contrast images.
Parasitophorous vacuoles are indicated by the arrowheads. 10D7
detected Irga6 on the PVM efficiently but the cytoplasmic, ER
membrane-associated Irga6 at a barely detectable level.

Colloidal Coomassie Staining—Gels were washed 30 min with
H₂O and subsequently placed in incubation solution (17% ammonium
sulfate (w/v), 20% MeOH, 2% phosphoric acid). After a 60-min incubation, solid
Coomassie Brilliant Blue G-250 (Serva) was added to the solution to a
concentration of 330 mg/500 ml and incubated 1-2 days. The gels were destained
by incubation in 20% MeOH for 1 min and stored in 5% acetic acid. All was done
at room temperature and while shaking.
Expression and Purification of Irga6 Proteins from E.
coli—pGEX-4T-2-Irga6 constructs were transformed into BL-21 E.
coli strain. Cells were grown at 37 °C to an A₆₀₀ of 0.8 when the expression of glutathione S-transferase-fused Irga6
proteins was induced by 0.1 mm isopropyl-β-d-thiogalactoside at 18 °C overnight. Cells
were harvested (5000 × g, 15 min, 4 °C); resuspended in
PBS, 2 mm DTT, Complete Mini protease inhibitor mixture without
EDTA (Roche Applied Science) and lysed using a microfluidizer (EmulsiFlex-C5;
Avestin) at a pressure of 150 megapascals. The lysates were cleared by
centrifugation at 50,000 × g for 60 min at 4 °C. The
soluble fraction was purified on a glutathione-Sepharose affinity column
(GSTrap FF 5 ml; Amersham Biosciences) equilibrated with PBS, 2 mm dithiothreitol. The glutathione S-transferase domain was cleaved off
by overnight incubation with thrombin (20 units/ml; Serva) on the resin at 4
°C. Free Irga6 was eluted with PBS, 2 mm dithiothreitol, and
the protein content in fractions was analyzed by SDS-PAGE and visualized by
Coomassie staining (22 (link)). The
protein-containing fractions were concentrated in a centrifugal concentrator
(Vivaspin20; Sartorius). Aliquots were shock-frozen in liquid nitrogen and
stored at -80 °C. The concentration of protein was determined by UV
spectrophotometry at 280 nm.