The preparation and partial purification of LpYhhN-GFP fusion protein from one liter of cells described above was scaled up to process protein from 3 liters of culture. The cells were lysed by French press, membranes were isolated by differential centrifugation and solubilized as described above. The 120,000 × g supernatant containing solubilized membranes was applied to a 5 ml nickel column and the fusion protein was eluted with an imidazole gradient. Three enriched 1.5 ml fractions from the nickel column were pooled and concentrated to 800 µl. For cleavage with TEV protease, the 4 ml reaction included 1.24 mg LpYhhN-GFP protein from the nickel column, 1.0 mM DTT, 0.1% DDM, 50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, and 1167 units of TEV protease (Invitrogen 12575-015). The reaction contents were gently mixed for 36 hours at 0 °C. Aliquots were retained for activity and gel analyses. The remainder (3500 µl) was loaded on to a one ml nickel column at 0.02 ml per minute, and the flow through containing un-tagged LpYhhN protein was concentrated. Aliquots were made 50% with glycerol and stored in small aliquots at −20 °C for kinetic analyses. Other aliquots were treated with 5 × loading buffer for analyses on 11% page gels. The GFP-His protein and the TEV protease (with His tag) were eluted late in a 20 ml gradient of 10 mM – 300 mM imidazole in buffer B.
Tev protease
Manufactured by Thermo Fisher Scientific
Sourced in United States
TEV protease is a site-specific cysteine protease enzyme derived from the Tobacco Etch Virus (TEV). It is commonly used in protein purification and processing applications to cleave fusion tags from recombinant proteins.
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Lab products found in correlation
Ni nta, by Qiagen (5 mentions)
Igg sepharose, by GE Healthcare (4 mentions)
Vivaspin concentrator, by Sartorius (3 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Glass bead, by Biospec (2 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (2 mentions)
Glutathione sepharose resin, by GE Healthcare (2 mentions)
Enrich sec 650, by Bio-Rad (2 mentions)
Igg sepharose bead slurry, by GE Healthcare (2 mentions)
Igg sepharose beads, by GE Healthcare (2 mentions)
3xflag peptide, by Merck Group (2 mentions)
Calmodulin affinity resin, by Agilent Technologies (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Ni nta, by Thermo Fisher Scientific (2 mentions)
Hiload 16 60 superdex 200, by GE Healthcare (2 mentions)
Amicon ultra 10 000 mwco centrifugal filter units, by Merck Group (2 mentions)
Glutathione sepharose bead, by Thermo Fisher Scientific (2 mentions)
Edtα free protease inhibitor mixture, by Roche (2 mentions)
Tev protease, by Qiagen (2 mentions)
Neutravidin agarose resin, by Thermo Fisher Scientific (2 mentions)
56 protocols using tev protease
1
Purification and Cleavage of LpYhhN-GFP Fusion Protein
2
Affinity Purification of Pre-60S Subunits
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Pre-60S subunit assembly intermediates were affinity-purified from whole-cell extracts with magnetic Dynabeads (Invitrogen) using TAP-tagged Nop7. Cell pellets were resuspended in RNP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.075% NP-40) and subjected to glass bead lysis. Lysates were incubated with IgG-coated Dynabeads for 1 h at 4°C with rocking, then the beads were washed three times with RNP buffer. Preribosomes were eluted from the Dynabeads by incubation at room temperature for 1 h with 10 U TEV protease (Invitrogen), which cleaves the TEV protease site within the TAP-tag. Preribosomes were precipitated with 10% TCA.
3
Germline Injections and TEV Protease Purification
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Germline injections were performed as described in Castellano-Pozo et al., 2020 (link) using a Narishige IM-31 pneumatic microinjector attached to an inverted Olympus IX71 microscope. Needles were made using a micropipette puller P-97 (Intracell) and borosilicate glass filaments with a 1.0 mm O.D. and 0.58 mm I.D. (BF100-58-10, Sutter Instruments). AcTEV protease (Thermo Fisher, Cat# 12575) was used in a mix containing 10 U/µl TEV protease in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 50% (v/v) glycerol, 0.1% (w/v) Triton X-100.
4
Recombinant Galectin-4 Expression and Purification
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Human recombinant gal 4 was produced at Protein Expertise Platform at Umeå University, was used. For this, Homo sapiens gal 4 (LGALS4; NM_006149.4) mRNA, cloned into plasmid pcDNA3.1-C-(k)DYK, was purchased (Genscript # OHu15835). An N-terminal Hisx6 +TEV-site was introduced by cloning LGALS4 into pET-His1a vector (NcoI + Acc65I). The cloning was done using Phusion polymerase (Thermo Fisher Scientific, # F631S), and the constructs were transformed into Bl21(DE3) competent cells. Cloning was confirmed by sanger sequencing (Eurofins Genomics). Expression was induced using Rosetta (DE3), auto-induction media, at 20°C for 16 hours. Bacterial cells were harvested, and lysis was performed by sonication (IKA-Werke GmbH). Protein was affinity purified using NiNTA (Thermo Fisher Scientific, # R90115), followed by tag removal using TEV-protease (Thermo Fisher Scientific, # 12575015). Both the Hisx6-TEV-tag and TEV-protease were removed by a second NiNTA run.
5
Purification of Biotin-Tagged Human Proteasomes
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The purification of Human proteasomes was performed from HEK293 cells expressing biotin-tagged human 4 subunit, as previously described [16 (link)]. The HEK293 cells were treated with 5 μg/ml of tunicamycin for 6 h. For pre-treatment experiments, the HEK293 cells were incubated for 10 h with 50 μM IU1, followed by treatment with 5 μg/ml of tunicamycin for 6 h. The cells were lysed in the buffer (50 mM NaH2PO4 pH 7.5; 5 mM MgCl2; 100 mM NaCl; 10% glycerol; 0.5% NP-40; 5 mM ATP; 1 mM DTT) containing protease inhibitor and homogenized with a Dounce homogenizer. The proteasome complex was isolated using agarose beads (Millipore, Billerica, MA, USA). The beads were then washed four times with lysis buffer and TEV buffer (50 mM Tris-HCl pH 7.5; 1 mM ATP; 10% glycerol). The proteasomes were eluted using TEV protease (Thermo Fisher, Waltham, MA, USA). The elutes were concentrated using Amicon ultra centrifugal filters (Millipore, Billerica, MA, USA).
6
Affinity Purification of Ribosome Intermediates
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Ribosome assembly intermediates were affinity-purified from whole-cell extracts with magnetic Dynabeads (Thermo Fisher Scientific), using TAP-tagged AFs Nop7 or Nog2. Cultures (250 mL) were grown either in galactose- or glucose-containing liquid media to an OD600 of 0.7–0.9. Cells were collected and resuspended in 3.5 mL of Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.075% detergent octylphenoxypolyethoxyethanol), and subjected to vortexing with glass beads (0.5 mm diameter, Biospec Products) eight times for 30 s, with incubation on ice in between vortexing. Extracts were clarified by centrifugation and bound to IgG-coated Dynabeads at 4 °C for 1 h. Beads were washed three times with the Lysis buffer and pre-ribosomes were eluted by cleaving the TEV Protease site within the TAP-tag, using 1–2 µL of TEV Protease (Thermo Fisher Scientific). Proteins were precipitated with 10% trichloroacetic acid (TCA), resuspended in SDS sample buffer and separated by SDS-PAGE on 4–20% Tris-Glycine Novex gels (Thermo Fisher Scientific).
7
Reconstitution of APC/C Ubiquitination
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Yeast cells carrying Cdc16-TAP and lacking Cdh1 were collected at OD600 = 1, flash frozen, and lysed using beat beating in lysis buffer. APC/C was immunopurified on IgG beads and activated by in vitro-translated Cdc20 or Cdh1. Cdc20 and Cdh1 used in Fig. 5e–g were expressed in insect cells using the baculovirus system and purified using Strep-tag-purification methods. For APC/C substrates, Pds1, Hsl1 fragment (aa 667–872), and Clb5 were C-terminally ZZ-tagged, cloned into plasmids under control of the T7 promoter, and translated in vitro using 35S-Methionine. Substrates were purified using IgG-coupled magnetic beads and cleaved with TEV protease (Thermo Fisher #12575015). E1 and E2 (Ubc4) were expressed in E. coli and purified60 (link),61 (link). E2 charging was performed in a reaction containing 0.2 mg/ml Uba1, 2 mg/ml Ubc4, 2 mg/ml methylated ubiquitin (Boston Biochem #U-501) and 1 mM ATP at 37 °C for 30 min. The ubiquitylation reaction was initiated by mixing activated APC/C, E2-ubiquitin conjugates, purified substrate, and buffer or indicated concentrations of single-stranded DNA at 25 °C. Reactions were terminated by addition of 2X SDS sample loading dye and separated by SDS–PAGE.
8
Mitochondrial Import and Crosslinking
9
Visualizing ORAI1 Protease Cleavage
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Control and NKD2 KO Jurkat T cells (3 × 106) were transfected with a plasmid encoding ORAI1N223A-TEV-pHluorin by electroporation with an ECM 830 electroporator. Twenty-four hours after transfection, cells were left untreated or were stimulated with soluble anti-CD3 antibody (10 μg/ml, OKT3) and cross-linked with anti-mouse secondary antibody for 20 minutes. Proteolytic cleavage was performed at room temperature (25°C) by adding 40 U TEV protease (Thermofisher) and 1 mM dithiothreitol (Thermofisher) for 20 min. The progress of cleavage was assayed by the loss of fluorescence using microscopy or flow cytometry.
10
Purification of Recombinant Transportin Protein
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BL21-Gold(DE3) cells transfected with pGEX4TT3-TEV-KapBeta273 were inoculated into 20 mL of LB + Amp and grown overnight at 37°C. The starter culture was transferred to 2 L of LB + Amp, grown to an OD600 of ~0.6 at 25°C, and then induced with 0.5 mM IPTG for 12 hours at 25°C. The cells were resuspended in 15 mL of transportin lysis buffer (50 mM Hepes, 150 mM NaCl, 15% glycerol, 2 mM dithiotheitol (DTT), 2 mM EDTA, pH 7.4 + PI) per liter of original culture, and then lysed as was done during the Ran purification. The cleared lysate was mixed with 500 μL glutathione Sepharose resin (GE Healthcare, #17-0756-01) and loaded onto a gravity column, which was washed with 20 mL of transportin lysis buffer, 25 mL of 50 mM Hepes, 100 mM NaCl, 1 mM EGTA, 10 mM MgOAc2, 2 mM DTT, 15% glycerol, 5 mM ATP, pH 7.4 + PI, and 25 mL of 20 mM Hepes, 20 mM NaCl, 2 mM EDTA, 10% glycerol, 2 mM DTT, pH 7.4. The GST tag was cleaved by incubating the Sepharose resin with 20 mM Hepes, 20 mM NaCl, 2 mM EDTA, 10% glycerol, 2 mM DTT, 0.5 units/μL TEV protease (ThermoFisher Scientific, #12575015), pH 7.4 for 2 h at room temperature, and the solution was then collected (0.5 mL fractions). The transportin protein was further purified by Enrich Sec 650 size exclusion chromatography (Biorad, #7801650) in 20 mM Hepes, 110 KOAc, 5 mM NaOAc, 2 mM MgOAc2, 2 mM DTT, pH 7.4.
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