Bl21 de3 cells

Recombinant proteins including: GST, GST-hTransportin, GST-xCrm1, GST-hExportin-t, 6xHis-hExportin-5, and GST-RanQ69L were expressed in BL21 (DE3) cells (New England BioLabs, C25271) by growing 1 L at 37⁰C for 3 hours or until the OD₆₀₀ was ~0.500. Protein expression was induced by adding IPTG (0.5 mL of 1 M stock) to a final concentration of 0.5 M and growing cells overnight at 16⁰C. The cells were collected by centrifugation, resuspended in 25 mL of bacterial lysis buffer (300 mM NaCl, 50 mM Tris, pH = 7.5) plus 5 mg lysozyme (BioPioneer, C0021) and frozen at −80⁰C. Cells were thawed on ice, then sonicated and centrifuged in an SS-34 rotor (Sorvall) to clear the lysate of insoluble material (24,000 G, 45 minutes, 4⁰C). The lysate was applied to Glutathione Agarose 4B beads (Prometheus Protein Biology Products, 20–542) or Super Ni-NTA Agarose Nickel beads (Lambda Biotech, G202) and the expressed proteins purified according to manufacturers’ instructions. Proteins were stored in aliquots at −80⁰C in Egg Lysis Buffer [ELB; 250 mM Sucrose, 2.5 mM MgCl₂, 50 mM KCl, 10 mM HEPES, and the pH was adjusted to 7.8 with KOH [47 (link)]]. Antibodies used in this study were generated from rabbit serum against Xenopus anti-Nup98 [51 (link)] and Xenopus anti-Nup133 [47 (link),50 (link)].

The S. pneumoniae LicA gene expressing sChok had been previously cloned into the pET28a plasmid [7 (link)]. This construct was generously provided to us by the group of Dr. Yuxing Chen. The plasmid was transformed into BL21(DE3) cells (New England Biolabs). These cells were then used to inoculate 10 mL of Luria Broth (LB) which was incubated overnight at 37 °C. The next morning, a 10 mL of fresh LB was inoculated to 2% with the overnight culture and then incubated at 37 °C until an O.D.₆₀₀ of 0.6 was reached. The culture was cooled on ice, and IPTG was added to a final concentration of 1 mM to induce production of sChok. The culture was then incubated overnight at room temperature after which it was centrifuged for 10 min at 3500 g, resuspended in 1 mL 100 mM Tris pH 8 and transferred to an Eppendorf tube. Three mg of Glasperlen beads (Sartorius Stedim) were added to the resuspension and lysis was performed using a Bead-Beater 16 (Biospec products). The lysate was centrifuged at 20,000 g for 30 min at 4 °C. The supernatant was removed, aliquoted into Eppendorf tubes, and stored at −20 °C. An aliquot was defrosted on ice when needed for use in an enzymatic reaction.

Plasmids pSMCAF015 and pSMCAF016 used for the expression of GFP and SpyCatcher-GFP from E. coli were assembled from existing constructs (pBAD-RFP and pBAD-SpyCatcher-RFP^{29 (link)}, respectively) by substituting the mRFP sequence with the GFP sequence (below). Similarly, plasmids pSMCAF032 and pSMCAF029 used for the expression of GDH and SpyCatcher-GSH from E. coli were assembled by introducing the GDH sequence in the same position. These plasmids were transformed into chemically competent BL21(DE3) cells (New England Biolabs – C2527H); single transformants were selected using ampicillin resistance.

To prepare biotinylated WT importin β-Avitag (pKW1982, pK1099), BL21DE3 cells (New England Biolabs, Ipswich, MA) were co-transfected with the respective plasmids together with pAC-biotin ligase (Avidity), followed by plating and growth in LB media containing ampicillin and chloramphenicol. After the 37°C cultures reached OD_600nm + 0.4–0.6, the cultures were cooled to room temperature, supplemented with 100 µM D-biotin, and the expression was induced with 0.3 mM IPTG at room temperature for 8–11 hr (pKW762). Proteins were purified on Ni-NTA resin as described for the non-biotinylated importin β fragments.