Pet 19b vector

All plasmids used in this study are listed in Supplementary file 1. Crm1 and Ran were cloned between the NdeI and XhoI sites in a pET19b vector (EMD Millipore, Darmstadt, Germany) that encodes an amino-terminal deca-histidine tag followed by a tobacco etch virus (TEV) protease site. Genes were amplified from their source vectors by polymerase chain reaction with phusion polymerase (New England Biolabs (NEB), Ipswich, MA) using forward and reverse primers (Integrated DNA technologies (IDT), Coralville, IA, listed in Supplementary file 2) containing NdeI or XhoI restriction sites, respectively. PCR products and the target vector were digested with restriction enzymes (NEB), purified (Qiagen, Venlo, Netherlands), ligated (Roche, Basel, Switzerland), and then transformed into E. coli DH5α. Plasmids from positive transformants were isolated (Qiagen), screened by restriction digests, and sequenced (University of California - Berkeley DNA Sequencing Facility, Berkeley, CA).

ORF of EaRFP cDNA were inserted into pET-19b vector (Novagen Merck KGaA, Darmstadt, Germany). E. coli BL21 (DE3) pLysS competent cells (Novagen) were used as a host strain for the recombinant construct and grown at 37 °C until reaching log phase. The expression of the fusion protein was then induced by the addition of 1 mM of isopropyl-β-D-thiogalactopyranoside, followed by culture at 28 °C for 16 h in LB medium. N-terminal His-tagged recombinant EaRFP was purified from the cell lysate by passing through an affinity column containing a high density of nickel-agarose beads (Agarose Bead Technologies, Madrid, Spain).