Affinity column

RNAs were prepared by in vitro transcription essentially as described [59 (link)]. The DNA template was plasmid DNA for E^ΔP5abc or a PCR product assembled from oligonucleotides for P5abc. P5abc constructs for DMS footprinting included 5′ and 3′ flanking hairpins for normalization and single-stranded regions to serve as markers or to anneal with a primer for reverse transcription and RNA isolation [42 (link)]. E^ΔP5abc mutants were generated by a Quikchange mutagenesis protocol (Agilent). Oligonucleotides for PCR assembly and mutagenesis were from IDT (San Diego, CA). DNA templates and RNA were isolated by affinity column (Qiagen). P5abc RNA was 5′-end labeled by treating it with shrimp alkaline phosphatase (New England Biolabs) followed by polynucleotide kinase and [γ-³²P]-ATP. Labeled RNA was purified by polyacrylamide gel electrophoresis, eluted into TE buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and stored at −20 °C.

DNA templates were prepared by assembly PCR with four partially overlapping oligonucleotides (Integrated DNA Technologies). Primer extension reactions were carried out using Phusion High-Fidelity DNA Polymerase (New England Bio-Labs) followed by purification of full-length, double-stranded DNA templates by Magnetic Bead PCR Clean-up (Axygen). PCR-amplified DNA template (0.2 μM) was used in transcription reactions with T7 RNA polymerase, as previously described (Cordero et al., 2014 (link)). RNA transcripts were isolated by affinity column (QIAGEN).

Full-length human PDE4D5 active mutant S126D and mutant S126D-F268A were subcloned into expression vector pGEX-6P-1 and expressed in E. coli BL21(DE3) cells (Invitrogen). Cells were cultured at 37°C in LB medium until optical density at 600 nm (OD₆₀₀) reached 0.6. Then, 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) was added to induce protein expression at 30°C for 4 hours. The PDE4D5 mutant proteins were purified using glutathione S-transferase bind resin (Millipore Sigma), glutathione elution, precision protease cleavage, and Superdex 200 (Millipore Sigma) columns. The purified proteins were dissolved in 50 mM tris-HCl buffer (pH 8.0) with 100 mM NaCl and 1 mM β-mercaptoethanol and stored at −80°C. The catalytic domain of human PDE4D5 (317–676) was subcloned into the expression vector pET15b and expressed in E. coli BL21(DE3) cells. Cells were cultured at 37°C until OD₆₀₀ reached 0.6, when 0.1 mM IPTG was added to induce protein expression at 15°C for 24 hours. The recombinant PDE4D5 catalytic domain was purified using Ni-NTA (nitrilotriacetic acid) affinity columns (Qiagen), thrombin cleavage, and Superdex 200 columns. PDE4D5 catalytic domain protein purity was greater than 95% as shown by SDS–polyacrylamide gel electrophoresis.