Pgex 6p 3 expression vector

To raise polyclonal antibodies against TMEM59L, a fusion protein containing glutathione S-transferase (GST) and the N-terminal region of TMEM59L was produced in E. coli. In brief, a Tmem59l cDNA fragment encoding amino acids 1–222 of TMEM59L was amplified by high fidelity PCR using Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA, USA). The cDNA fragment was inserted into the pGEX-6P-3 expression vector (GE Healthcare, Buckinghamshire, UK), and the resulting plasmid was introduced into E. coli to produce the GST-TMEM59L fusion protein, which was purified using a Glutathione Sepharose 4B (GE Healthcare) column. The purified fusion protein was used to generate anti-TMEM59L polyclonal antisera in rabbits. The TMEM59L immunoreactive antiserum was immunoaffinity-purified, and the resulting purified antibody was used as described below.

The expression plasmids for the fusion proteins, TAT-EGFP-peptide 1 (STLTASEV) and TAT-EGFP-scramble 1 (EASTSLVT) were prepared by the site-directed mutagenesis of DNA sequences encoding TAT-EGFP cloned in a pGEX-6P-3 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) (Kizaka-Kondoh et al., 2009 (link)). DNA primers for the amplification of plasmids were as follows: for TAT-EGFP-peptide 1, 5′-GCCAGCGAGGTGTAAATCGTGACTGACTGACGATCTGCC-3′ and 5′-GGTCAGGGTGCTGCCCTTGTACAGCTCGTCCATGGCG-3′; for TAT-EGFP-scramble 1, 5′-AGCCTGGTGACCTAAATCGTGACTGACTGACGATCTGCC-3′ and 5′-GGTGCTGGCCTCGCCCTTGTACAGCTCGTCCATGGCG-3′. The resultant plasmids were introduced into BL21-CodonPlus (DE3) cells (Agilent Technologies, Santa Clara, CA, USA). Fusion proteins were expressed as glutathione S-transferase (GST)-tagged proteins and purified by affinity chromatography, as previously described (Kizaka-Kondoh et al., 2009 (link)). The GST-tag was removed, and final proteins were equilibrated in PBS.