M9 minimal medium

PP2Aa_9–589, FAM122A_1–124 (FAM122A_Nterm), FAM122A_29–120 (FAM122A_ID), FAM122A_67–120, ARPP19, ARPP19_S104A, ARPP19_19–75 and p107_612–687 were subcloned into pTHMT containing an N-terminal His₆-tag followed by maltose binding protein (MBP) and a tobacco etch virus (TEV) protease cleavage site. For expression, plasmid DNAs were transformed into Escherichia coli BL21 (DE3) RIL or BL21 (DE3) cells (Agilent). Freshly transformed cells were grown at 37 °C in LB broth containing kanamycin antibiotics (50 µg ml⁻¹) until they reached an optical density (OD₆₀₀) of ~0.8. Protein expression was induced by addition of 1 mM β-d-thiogalactopyranoside (IPTG) to the culture medium, and cultures were allowed to grow overnight (18–20 h, 250 rpm shaking) at 18 °C. Cells were collected by centrifugation (8,000g, 15 min, 4 °C) and stored at −80 °C until purification. Expression of uniformly ¹³C- and/or ¹⁵N-labelled protein was carried out by growing freshly transformed cells in M9 minimal medium containing 4 g l⁻¹ [¹³C]-d-glucose and/or 1 g l⁻¹¹⁵NH₄Cl (Cambridge Isotopes Laboratories) as the sole carbon and nitrogen sources, respectively. FAM122A_ID variants E92K, R105L, V107G, S120C, E104A/S120C, E106A/S120C, R84A/L85A/S120C, I88A/K89A/S120C, E91K/S120C, E92K/S120C, FAM_67-120S120C and ARPP19/S10C were generated by site-directed mutagenesis, sequence verified and expressed as described above.

For specific spin labeling at residue 27, we generated the triple-mutation construct, H27C/C57S/C113D-Pin1 (3m-Pin1). The C57S and C113D substitutions were to prevent off-target spin labeling. Cys⁵⁷ is largely surface-exposed, and the choice of C57S was based on side-chain similarity. Cys¹¹³ is part of the substrate proline-binding pocket. We chose C113D based on previous studies demonstrating that C113D-Pin1 maintains activity in vivo and in vitro (29 (link)).
Both H27C/C57S/C113D-Pin1 (3m-Pin1) and WT-Pin1 were overexpressed in Escherichia coli BL21 (DE3) cells (Novagen). Cells were first grown at 37 °C in lysogeny broth medium until they reached an A₆₀₀ of 0.8–1.0. For isotope-enriched protein, cells were harvested and resuspended in M9 minimal medium containing ¹⁵NH₄Cl and/or [¹³C]glucose (Cambridge Isotope Laboratories) as the sole nitrogen and carbon sources (48 (link)). Overexpression of 3m-Pin1 was induced by adding 1 mm isopropyl β-d-1-thiogalactopyranoside and incubated at 16 °C (to slow expression and allow proper folding) for ∼20 h. Overexpression of WT-Pin1 was induced at 26 °C for ∼16 h. Cells expressing protein were harvested and resuspended in 50 mm HEPES buffer (pH 7.5 for WT-Pin1 and pH 6.5 for 3m-Pin1 containing 1 mm EDTA). Both 3m- and WT-Pin1 constructs were purified using a HiTrap SP column followed by size exclusion (HiPrep Sephacryl S-200 HR).

To express the Trx protein, the pET32a-Trx gene was transformed into E. coli Rosetta-gami B to generate E. coli/pET32a-Trx. The E. coli cells obtained in this way were then allowed to grow at 37 °C until the OD₆₀₀ was approximately 0.6. After decreasing the temperature to 18 °C, the protein was induced by the addition of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells were grown for an additional 22 h at 18 °C and purified via His-tag affinity followed by thrombin cleavage and size-exclusion chromatography. For ¹⁵N-labeled Trx NMR samples, the E. coli cells were grown in an M9 minimal medium supplemented with ¹⁵NH₄Cl as the sole nitrogen source (99% ¹⁵N; Cambridge Isotope Laboratories, Inc.). Cell lysates were purified using a His-tag column followed by gel filtration (HiLoad 16/600 Superdex 75 pg, GE Healthcare, Uppsala, Sweden) equilibrated with 20 mM HEPES (pH 7.5), 150 mM NaCl and 2 mM dithiothreitol (DTT).