Protein purification and fluorescent labeling

Skeletal actin was prepared from acetone powder of rabbit skeletal muscle (Pel-Freez Biologicals, Rogers, AR) as previously described using G-buffer (5 mM tris-HCl, 0.2 mM CaCl₂, 0.2 mM ATP, and 5 mM β-mercaptoethanol) and multiple rounds of polymerization and depolymerization (71). Alexa Fluor 488– and TMR (Molecular Probes/Thermo Fisher Scientific, Waltham, MA)–labeled actin samples were prepared from G-actin in G-buffer devoid of reducing agents as previously described (45). N-(1-pyrenyl)iodoacetamide (pyrene; Thermo Fisher Scientific, Waltham, MA)–labeled actin was prepared from F-actin followed by depolymerization upon dialysis in G-buffer as described (72, 73). Labeled and unlabeled G-actin was further purified by size exclusion chromatography (Sephacryl S200-HR; Cytiva/GE Healthcare, Marlborough, MA). Unlabeled actin was stored on ice in G-buffer containing β-mercaptoethanol and used within 4 weeks with a dialysis to G-buffer after 2 weeks of storage. Labeled G-actin was snap-frozen and stored at −80°C.

Recombinant proteins were expressed in BL21-CodonPlus(DE3)-RP E. coli (Agilent Technologies, Santa Clara, CA) unless indicated otherwise. Human PFN1 was purified as previously described (74). PFN1 was bound to a poly-l-proline sepharose resin, eluted under denaturing conditions, and dialyzed against three buffer changes of storage buffer [2 mM tris-HCl (pH 8.0), 0.2 mM EGTA, 1 mM dithiothreitol (DTT), and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)].

ΔNS-VopF was subcloned downstream of the SNAP26b gene in pSNAP-tag(T7)-2 vector (New England Biolabs) in-frame with 6xHis-tag using Q5 High-Fidelity DNA Polymerase (New England Biolabs) to amplify cDNA and NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA). 6xHis-SNAP-tagged ΔNS-VopF was purified using TALON Metal Affinity Resin (Takara Bio USA, San Jose, CA) according to the manufacturer’s instructions. For labeling, SNAP-Surface-549 or SNAP-biotin (New England Biolabs, Ipswich, MA) were added at 2-mole excess in a buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM DTT, and 0.1 mM PMSF overnight at 4°C, followed by gel filtration or dialysis to remove free dye.

His-tagged mouse CP heterodimer (α1 and β2 subunits in pRSFDuet, a gift from J. Cooper) was purified as described previously (66) using TALON Metal Affinity Resin (Takara Bio USA, San Jose, CA) followed by a ceramic hydroxyapatite column (40 μm; EconoFit CHT type I, Bio-Rad Laboratories, Hercules, CA) and gel filtration (Sephacryl S-200 HR, Cytiva/GE Healthcare, Marlborough, MA). The purified CP was stored in 20 mM Mops, 100 mM KCl, and 1 mM tris(2-carboxyethyl)phosphine (TCEP) (pH 7.2) at −80°C.

SNAP-tagged CP was expressed in E. coli BL21 DE3 (Gold Biotechnology Inc., Olivette, MO) by growing cells to log phase at 37°C in terrific broth medium and then inducing expression using 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 18°C overnight. Cells were harvested by centrifugation, and pellets were stored at −80°C. Frozen pellets were resuspended in lysis buffer [20 mM NaPO₄ (pH 7.8), 300 mM NaCl, 1 mM DTT, 15 mM imidazole, and 1 mM PMSF] supplemented with a protease inhibitor cocktail (0.5 μM each of pepstatin A, antipain, leupeptin, aprotinin, and chymostatin). Cells were lysed by sonication with a tip sonicator while keeping the tubes on ice. The lysate was cleared by centrifugation at 150,000g for 30 min at 4°C. The supernatant was incubated with Ni–nitrilotriacetic acid agarose beads (Qiagen, Hilden, Germany) for 2 hours at 4°C, followed by extensive washing of the beads with washing buffer [20 mM NaPO₄ (pH 7.8), 300 mM NaCl, 1 mM DTT, and 25 mM imidazole] to remove nonspecifically bound proteins. SNAP-CP was eluted using 20 mM NaPO₄ (pH 7.8), 300 mM NaCl, 250 mM imidazole, and 1 mM DTT. The eluted protein was concentrated and labeled with SNAP-Surface 549 or SNAP-biotin (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions. Free dye was removed using size exclusion chromatography by loading the labeled protein on a Superdex 200 Increase 10/300 GL gel filtration column (Cytiva/GE Healthcare, Marlborough, MA) eluted with 20 mM Hepes (pH 7.5), 150 mM KCl, and 0.5 mM DTT. Fractions containing the protein were combined. Protein concentration and labeling fraction were determined by measuring the absorbance at 280 and 560 nm (ε₂₈₀ = 102,165 M⁻¹ cm⁻¹ and ε₅₆₀ = 140,300 M⁻¹ cm⁻¹). Purified protein was aliquoted, snap-frozen in liquid N₂, and stored at −80°C.

Protocatechuate 3,4-dioxygenase (PCD) from Pseudomonas putida was expressed from pVP91A-pcaHG plasmid [RRID:Addgene_113766 (75)] in Rosetta(DE3) E. coli cells (MilliporeSigma, Burlington, MA) in LB media supplemented with 0.5 mM MgSO₄ and 1 mM CaCl₂. Protein expression was induced by addition of 250 μM IPTG, and the culture was supplemented with 25 μM ferrous ammonium sulfate and grown overnight at 15°C. Cells were resuspended in 50 mM sodium phosphate buffer (pH 8.0) containing 150 mM NaCl, 25 μM ferrous ammonium sulfate, and 1 mM PMSF and lysed by French press. PCD was purified using TALON Metal Affinity Resin (Takara Bio USA, San Jose, CA) followed by size exclusion chromatography. Purified PCD was stored in 50% glycerol, 10 mM Hepes (pH 8.0), 75 mM NaCl, and 0.05 mM PMSF at −80°C.