Protein expression and purification

For structure determination, the DNA encoding the RRM1 (Pro5–Leu96) of human SF3b49 (GenBank: {"type":"entrez-protein","attrs":{"text":"BAD97042.1","term_id":"62898205","term_text":"BAD97042.1"}}BAD97042.1) was subcloned by PCR from the human cDNA clone with the ID RIKEN cDNA hss001003928. Note that the amino acid sequence of the expressed RRM protein differs only in position 75 (Asn vs. Asp) from that of the protein of SwissProt accession no. {"type":"entrez-protein","attrs":{"text":"Q15427","term_id":"2500587","term_text":"Q15427"}}Q15427. The changed position is located at the C‐terminal end of α2, which is located in a side nearly opposite from the interaction site in the helical region.

The DNA fragment was cloned into the expression vector pCR2.1 (Invitrogen, Carlsbad, CA) as a fusion with an N‐terminal native His affinity tag and a TEV protease cleavage site. The ¹⁵N, ¹³C ‐labeled fusion protein was synthesized using a cell‐free protein expression system from E. coli.34, 35 The resulting tagged protein was purified by a 5‐ml His Trap column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) with an imidazole gradient from 12 to 500 mM. After tag removal, the tag‐free protein was further purified by HiTrap SP and HiTrap Q column chromatography (GE Healthcare).

For the NMR titration experiments and the pull‐down assay, the RRM1 (Pro5–Leu96) of human SF3b49 was cloned into the Nde I/Bam HI sites of pET‐15b (Novagen). Each of the fragments of SF3b145 ({"type":"entrez-protein","attrs":{"text":"Q13435","term_id":"296452908","term_text":"Q13435"}}Q13435, 553–631, and 598–631), was cloned into the Eco RI/Sal I sites of pGEX6P‐1 (GE Healthcare). In all constructs, a TEV protease cleavage site was placed between the tag and the protein sequences. E. coli strain BL21 (DE3) cells with the recombinant plasmids were grown at 37°C in LB medium supplemented with 50 mg/L of ampicillin for the non‐labeled samples and in modified minimal medium36 supplemented with 50 mg/L ampicillin for the ¹⁵N, ¹³C‐labeled samples. After isopropyl β‐D‐1‐thiogalactopyranoside induction (1 mM), the harvested cells were lyzed, and the lysates were applied to a His Accept column (Nacalai Tesque) or a Glutathione Sepharose 4 Fast Flow column (GE Healthcare) eluted by the addition of imidazole or glutathione. The tag‐free SF3b49 RRM1 was further purified by RESOURCE Q column chromatography (GE Healthcare). The GST‐tagged SF3b145(553–631) and the tag‐free SF3b145(598–631) were further purified by gel‐filtration column chromatography (GE Healthcare). The peptides corresponding to the two non‐labeled SF3b145 fragments [SF3b145(553–597) and SF3b145(598–631)] were purchased from Toray Research Center.