Pmal c

IMP3 RRM12 (residues 1–161) and the Y5A mutant were cloned by PCR into a derivative of pMalc (New England BioLabs) that contains a Tobacco Etch virus (TEV) protease site after the maltose-binding protein (MBP) tag. A C-terminal His₆ tag was added by PCR to ensure purification of full-length fusion proteins. The constructs were transformed into Escherichia coli strain Rosetta² (EMD Bio-sciences), and recombinant protein was induced with 1 mM IPTG for 4 h at 37°C. Cell pellets were resuspended in lysis buffer (50 mM Tris pH 7.5, 1.5 M NaCl, 1 mM EDTA, 1 mM DTT) supplemented with one Complete EDTA-free protease inhibitor tablet (Roche), and were lysed by sonication. Cell debris was removed by centrifugation, and the soluble fusion protein was purified by amylose affinity chromatography (New England BioLabs) followed by Ni-NTA affinity chromatography and size exclusion column (SEC) S200 (GE Healthcare). The protein concentrations were calculated by measuring the absorbance at 280 nm and using extinction coefficients determined by ProtParam (Gasteiger et al. 2005 ).

For transfection, the BglII–EcoRI fragment of CBP80 NLS (CYMSRRRHSDENDGGQPHKRRKTSDANETED; Miyamoto et al., 1997 (link)) and the BamHI–EcoRI fragments of SV NLS (PKKKRKV; Kalderon et al., 1984 (link)), hPY-NLS of hnRNP A1 (hM9NLS; GGSYNDFGNYNNQSSNFGPMKGGNFGGRSSGPYGGGGQY; Cansizoglu et al., 2007 (link)) and HIV-1 Rev NES (DVSDLPPLERLTLRGG; Güttler et al., 2010 (link)) were inserted into the BglII and EcoRI sites of mammalian expression vector pEGFP-C1 (6086-1; Takara Bio Inc.).
For recombinant protein purification, the following constructs were used: GST-SV NLS–GFP, Flag-Ran (Q69L), and Ran (human) were generated by inserting BamHI–EcoRI fragments into BamHI–EcoRI sites of the Escherichia coli N-terminal GST-fused protein expression vector pGEX-2T (GE Healthcare). Imp α1 (human), Imp β (human), and CAS (human) were generated by inserting BamHI–XhoI fragments into the BamHI and XhoI sites of the E. coli GST-fused protein expression vector pGEX-6P-1 (GE Healthcare). The pMALx2-C vector for expression of MBPx2 was generated by inserting EcoRI–HindIII PCR fragment of MBP into EcoRI and HindIII sites of E. coli MBP-fused protein expression vector pMAL-C (New England Biolabs, Inc.). Then, BamHI–EcoRI fragments of the SV NLS BglII–EcoRI fragment of CBP80 NLS were inserted into BamHI and EcoRI sites of pMALx2-C.