Pgex 6p 2 plasmid

GABARAPL1 full-length or GABARAP chimera sequences were inserted between the BamHI and NotI sites of a pGEX-6P2 plasmid (GE Healthcare, 28–9546-50) containing a cleavable glutathione S-transferase tag. Protein expression was performed at 30°C in E. coli Rosetta (DE3) pLysS (Novagen, 71403). Bacteria were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 0.1% TX-100, 0.5 mM TCEP [Melford Laboratories, T2650], 0.5 mM AEBSF [AppliChem, A1421], 15 μg/ml benzamidine [Melford Laboratories, B4101]). The fusion protein was batch-adsorbed onto a glutathione-Sepharose affinity matrix and GABARAPL1 recovered by cleavage with 3C protease (made in house) at 4°C overnight in 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM TCEP. The protein was then purified by size exclusion chromatography using a Superdex 75 column equilibrated and run in 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5 mM TCEP. Peptides were synthesized by the Francis Crick Institute Peptide Chemistry Science Technology Platform.

Amplification of Cpn 0810 was performed using polymerase chain reaction (PCR), based on the following primer pairs: P1, 5′-CGCGGATCCATGAATAAAAAGCCCAAGAAAAC-3′, and P2, 5′-TTTTCCTTTTGCGGCCGCTTACTCAGC GCCTTTAACCAT-3′.
Amplification was performed in a final reaction volume of 50 μl, containing 39.6 μl ddH₂O, 5 μl 10X Pfu buffer, 1 μl dNTP mix (10mM), 1 μl P1 primer, 1 μl P2 primer, 0.4 μl DNA Polymerase (5 units) and 2 μl Cpn templates. The amplification conditions were as follows: Initial polymerase activation at 94°C (5 min); 30 cycles of 94°C (30 sec), 52°C (45 sec) and 72°C (3 min); and a final elongation step at 72°C for 10 min. Distilled water was used as a negative control. The amplification products (363 bp) were subjected to 1.0% agarose gel electrophoresis containing ethidium bromide.
The PCR products were digested with BamHI and NotI (Promega Corporation, Madison, WI, USA), and ligated into the pGEX6p-2 plasmid (GE Healthcare, Piscataway, NJ, USA). The recombinant plasmid was transformed into E. coli BL21 competent cells, and the positive clones were screened by PCR and sequencing.

The Exosc3 open reading frame encoding the mouse EXOSC3 protein was cloned into pGEX-6P-2 plasmid (GE Healthcare Life Sciences [now Cytiva]) to create an N terminally GST-tagged EXOSC3 construct. Recombinant GST-EXOSC3 was expressed in Escherichia coli Rosetta 2 (DE3). The GST fusion protein was purified by affinity chromatography on GSH-Sepharose (GE Healthcare Life Sciences [now Cytiva]) in 300 mM NaCl, 50 mM Hepes/NaOH (pH 7.5), 5% glycerol and 2 mM beta-mercapto-ethanol. The fusion protein was eluted by addition of 30 mM reduced GSH. The GST tag was removed using PreScission protease (GE Healthcare Life Sciences [now Cytiva]), and the EXOSC3 protein was further purified by collecting the flowthrough of a second affinity chromatography on GSH-Sepharose resin. The untagged protein was further purified on a Superdex200 size-exclusion chromatography column equilibrated in 150 mM NaCl, 30 mM Hepes/NaOH (pH 7.5), 5% glycerol. Expression and purification of both recombinant GST-EXOSC3 and untagged EXOSC3 were confirmed by SDS-PAGE, followed by Coomassie staining. The purified untagged protein was used as an immunogen to raise rabbit polyclonal antibodies by Josman, LLC. Sera containing anti-EXOSC3 antibodies was collected 21 days after immunization and used directly for immunoblotting, immunoprecipitation, and immunofluorescence.