Recombinant Protein Expression and Purification

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)].