Escherichia coli bl21 codonplus de3 ril cells

SF3A1-UBL (residues 704 to 793) was cloned into pET24b (Novagen) in fusion with an N-terminal GB1 solubility tag and a 6×His tag cleavable by tobacco etch virus (TEV) protease (pET24-GB1-TEV-UBL). Mutants were generated by site-directed mutagenesis using the quick-change protocol and specific primers listed in SI Appendix, Table S3. All plasmids were sequenced and transformed into Escherichia coli BL21‐Codon Plus (DE3)‐RIL cells (Agilent Technologies) for protein expression (detailed expression protocol can be found in SI Appendix). The protein was purified by Ni-affinity chromatography and size exclusion chromatography in the NMR buffer [10 mM sodium phosphate (pH 6), 50 mM NaCl] (see SI Appendix for a detailed purification protocol). Final protein purity was checked by sodium dodecyl sulfate gels and analyzed for nucleic acid contamination using A_260nm/A_280nm. Protein concentration was estimated using A_280nm by calculating with the theoretical extinction coefficient of 9,970 M⁻¹ cm⁻¹ and stored at −80 °C. All point mutants were produced using the same protocol, and their correct folding was assessed by recording a 1D ¹H NMR spectrum (SI Appendix, Fig. S3). GST-SF3A1-UBL for EMSA experiments was prepared as before (16 (link)). U1 snRNP in vitro reconstitution was performed as described previously (21 (link)).