Primer sequences used here will be provided upon request. Deletion fragments of CD95, Plk3 and procaspase-8 were generated by standard techniques. Site-directed mutagenesis was performed using the QuikChange protocol and PfuUltra II Fusion HS DNA polymerase (Stratagene). Procaspase-8-WT, its subfragments (NT, p18, p10) and its mutants were inserted into the HindIII/XbaI sites of the pGEX 5X-3 vector (GE Healthcare). 3 × Flag-tagged procaspase-8-WT and its mutants (T273A, -T273D) were inserted into the BamHI/XbaI sites of the pcDNA3.1-V5 vector (Invitrogen). Plk3-WT and its subfragments were inserted into the BamHI and EcoRI sites of the Myc-, V5- or 3xFlag-tagged pcDNA3.1-Hygro+ vector (Invitrogen) and the pGEX 5X-3 vector. All constructs were confirmed by sequencing.
Pgex 5x 3 vector
Manufactured by GE Healthcare
The PGEX-5X-3 vector is a bacterial expression vector used for the production of recombinant proteins in Escherichia coli. It contains a glutathione S-transferase (GST) tag sequence, allowing for affinity purification of the expressed protein. The vector also includes a tac promoter, which enables inducible expression of the target gene.
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Lab products found in correlation
Pcdna3.1 hygro vector, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 v5 vector, by Thermo Fisher Scientific (1 mentions)
Pjet1, by Thermo Fisher Scientific (1 mentions)
Primestar max polymerase, by Takara Bio (1 mentions)
Bl21 de3 plyse, by Thermo Fisher Scientific (1 mentions)
Gst gene fusion system, by GE Healthcare (1 mentions)
Bl21 de3 plys, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose resin, by Qiagen (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Factor xa, by GE Healthcare (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
Pgex 6p 3 vector, by GE Healthcare (1 mentions)
8 protocols using pgex 5x 3 vector
1
Generating Caspase-8, Plk3, and CD95 Constructs
2
Molecular Cloning and Mutagenesis of MAPK Pathway
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Coding regions of MPK3, MPK4, and MPK6 were amplified by PCR, ligated in frame into pGEX5x-3 vector (GE Healthcare) and sequenced. MKK1, MKK2, MKK4, MKK5, MKS1, VQ4 and AT1G78150.1 were cloned into pETλHIS [22 (link)] using restriction enzymes listed in Additional file 1 : Table S1 and transformed into E. coli BL21. Coding regions of the MPK substrates AT2G26530, AT1G78150, AT4G38710, and AT3G11330 were amplified by PCR and cloned into pJET1.2 (Thermo Scientific). Site-directed mutagenesis (Additional file 1 : Table S2) was performed either by double joint PCR as described [23 (link)] or using the QuickChangeII site-directed mutagenesis kit (Stratagene).
3
Botulinum Hemagglutinin Genes Cloning
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DNA fragments encoding botulinum hemagglutinins were amplified by PCR from genomic DNA isolated from C. botulinum type B strain Okra or type C strain Stockholm using gene-specific primers containing restriction sites at their 5′ ends. The type B HA1 and HA2 fragments were inserted into the HindIII-KpnI site of the pT7-FLAG-1 vector (Sigma Aldrich). The type C HA1 fragment was inserted into the NotI-BglII site of the pT7-FLAG-1 vector. The HA3 fragments were inserted into the KpnI-SalI site of the pET52b(+) expression vector (Merck Millipore). The type B HA1 and HA3 fragments were also inserted into the BamHI-SalI site of the pGEX-5X-3 vector (GE Healthcare). Site-directed mutagenesis was performed using the PrimeSTAR Max polymerase (Takara Bio). The inserted regions of these vectors were confirmed by DNA sequencing.
4
Recombinant Expression of Enolase (EF1961) in E. coli
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EndoE, EndoE(E186Q) and EndoE(E662Q) were recombinantly expressed in E. coli as previously described using the GST gene fusion system from GE Healthcare [22] . Recombinant expression of the enolase (EF1961) was performed using the same protocol. Briefly, a 1299 bp fragment of EF1961 was amplified using the following primers: 5′-CGGGATCC ACATGTCAATTATTACTGATA-3′ with a BamHI site (underlined) and 5′-CCCTCGAG TTATTTGTTTTTTAAGTTG-3′ with an XhoI site (underlined). The PCR fragment was cloned into the pGEX-5x-3 vector (GE Healthcare) and transformed into the E. coli BL21 (DE3) pLysE (Invitrogen) strain for expression of EF1961.
5
Recombinant Protein Production and Purification
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TRIM32-HIS was expressed from pET28b vector (Clontech Laboratories, Mountain View, CA) and PR8-PB1-GST was expressed from pGEX-5X-3 vector (GE Healthcare Life Sciences, Pittsburgh, PA) in Escherichia coli BL21(DE3) pLysS (Life Technologies) induced with 0.2 mM isopropyl-1-thio-β-D-galactopyranoside and 200 μM ZnSO4 for 16 hr at 18°C as detailed elsewhere [38 (link)]. GST-tagged proteins were purified with glutathione Sepharose 4B beads according to the manufacturer’s protocol (GE Healthcare Life Sciences). HIS-tag proteins were purified with Ni-NTA agarose resins according to the manufacturer’s protocol (Qiagen).
6
Recombinant GST-ZNF32 Protein Expression
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Full-length ZNF32 was cloned into the pGEX-5X-3 vector (GE Healthcare, United Kindom) and transformed into E.coli BL21(DE3) star competent cells. The fusion protein was expressed under optimum folding conditions [46 (link)], and protein samples were collected at each step for 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were visualized with Coomassie brilliant blue R 250, and the expression level of the fusion protein was estimated by densitometric analysis with Quantity One 1D image analysis (Bio-Rad).
GST-ZNF32 fusion protein from the supernatant was applied to affinity chromatography with Glutathione Sepharose 4B (GE Healthcare, United Kindom) according to the manufacturer's instructions. Purified protein was digested with Factor Xa (GE Healthcare, United Kindom) for 16 hrs in 1 mM CaCl2, 100 mM NaCl, and 50 mM Tri-HCl (pH 8.0) at 22°C to remove the GST tag. Protein samples were used for Coomassie brilliant blue R 250 Staining and western blotting.
GST-ZNF32 fusion protein from the supernatant was applied to affinity chromatography with Glutathione Sepharose 4B (GE Healthcare, United Kindom) according to the manufacturer's instructions. Purified protein was digested with Factor Xa (GE Healthcare, United Kindom) for 16 hrs in 1 mM CaCl2, 100 mM NaCl, and 50 mM Tri-HCl (pH 8.0) at 22°C to remove the GST tag. Protein samples were used for Coomassie brilliant blue R 250 Staining and western blotting.
7
Construction of GST-Fusion Proteins
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To construct GST-Mybl1 and GST-Dmrtc2, cDNA fragments were amplified by PCR using human Mybl1 cDNA and mouse Dmrtc2 cDNA (from Dr. D. Zarkower) as templates and subcloned into pGEX-5X-3 vector (GE Healthcare). GST-Mybl1-N and GST-Mybl1-C contain DNA segments encoding N-terminal (aa 1–376) and C-terminal (aa 376–752) fragments of Mybl1, respectively. GST-Dmrtc2-N contains a DNA segment encoding N-terminal fragment (aa 1–201) of Dmrtc2. To construct GST-Lin9, -Lin37, -Lin52 and -Lin54, corresponding full-length human cDNAs were subcloned into pGEX-6P-3 vector (GE Healthcare). The GST-containing constructs were expressed in E. Coli BL21, and proteins were purified using glutathione sepharose (GE Healthcare).
8
Recombinant Lubricin and COMP Fragments
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RC human lubricin with a FLAG-tag and a truncated mucin-like domain (without amino acids (AA) 403-870) was produced in 293F cells using p3xFLAG-CMV-8 vector (SigmaeAldrich) and purified on anti-FLAG beads. The same method was used to produce four lubricin fragments named L25-221 (all molecular weights are calculated, MW 21.9 kDa), L220-402 (MW 19.2 kDa), L871-1078 (MW 22.6 kDa), L1079-1404 (MW 37.2 kDa, Fig. 2(A)). GST-tagged fragments of the N-terminal domain divided by exon boundaries (exons 2e5), named L25-72 (MW 5.3 kDa), L67-109 (MW 5.0 kDa), L105-160 (MW 6.0 kDa), L155-202 (MW 5.2 kDa), were produced using pGEX-5X-3 vector (GE Healthcare) and Rosetta 2 E. coli (Novagen), purified in native conditions using glutathione beads (Pierce). The His-tagged fragments of COMP [Fig. 2(A)] named mCOMP (AA 73-757, MW 79.3 kDa), TII1-TIII8 (AA 73-517, MW 51.7 kDa), TIII1-CG (AA 268-757, MW 59.0 kDa), TIII1-8 (AA 268-517, MW 31.5 kDa) and CG (AA 518-757, MW 31.9 kDa), based on the AA sequence of the human COMP reference sequence (NM_000095.2), were produced in human 293-EBNA cells and purified as previously described 21 . The identity of the proteins was confirmed by mass spectrometry, and purity by SDS-PAGE (Fig. S3).
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