Pcold 2 dna

Human ERα, importin-α1, importin-α2, importin-α4, importin-β1 and TNPO2 were generated by polymerase chain reaction (PCR) amplification from total RNA from MCF-7 and HeLa cells using appropriate primers (Supplementary Table ), and subsequently subcloned into the pENTR vector (Thermo Fisher Scientific, Waltham, MA, USA). ERα mutants and importin-α ΔIBB were constructed by site-dir1ected mutagenesis and by inverse PCR mutagenesis with the KOD-Plus-Neo, Ligation high Ver.2, and T4 Polynucleotide Kinase enzymes (KOD-401, LGK-201, PNK-111, TOYOBO, Osaka, Japan) using primers (Supplementary Table 1). Then, using the Gateway system (Thermo Fisher Scientific), the genes were subcloned into p3 × FLAG-CMV DEST^{61 (link)}, pcDNA3.1/Zeo(+)-EGFP DEST^{62 (link)}, and pcDNA3.1/Zeo(+) DEST, pGEX-6P-2 DEST, which were generated by a method similar to^{62 (link),63 (link)} using pcDNA3.1/Zeo(+) (Thermo Fisher Scientific), pGEX-6P-2 (Cytiva, Piscataway, NJ, USA) and the Reading Frame Cassette of the Gateway Conversion System (Thermo Fisher Scientific). pCold II Ran Q69L was constructed by inserting the human Ran Q69L mutant into pCold II DNA (Takara Bio Inc., Shiga, Japan).

The cDNA sequence of MjGCTL was amplified using rMjGCTL primers (Table 1), where EcoRI and NotI sites were added to the forward and reverse primers (5′ and 3′ end), respectively. The rMjGCTL PCR was cloned then cloned into the restriction sites of expression vector pMT:BiP:V5-His C (Invitrogen, Carlsbad, CA, USA). Recombinant plasmid was then transfected using Effectene Transfection Reagent (Qiagen, Germany) following manufacturer’s instructions. Stable cell lines were selected by passaging the cells several times on SDM with 125 mg/ml of blasticidin. Following the large-scale production of the stable cell-lines, protein expression was then induced by CuSO₄ (600 μM). One day after induction, the cells were centrifuged and rMjGCTL was purified from the supernatant using Ni-NTA agarose (Qiagen) purification column with 500 mM imidazole. The purified protein was quantified using DC protein assay (BIO-RAD) following the manufacturer’s protocol.
Prior to using S2 cells, production of rMjGCTL through bacterial cell system was also attempted through cloning MjGCTL to pCold II DNA (Takara bio, Japan) and transforming it to Escherichia coli (Origami B(DE3)), however the purification of the recombinant protein from large-scale production was unsuccessful.