2.1. Expression and purification of recombinant proteins

Human Them1 consists of two splice variants (Them1a and Them1b) that are distinguished by an additional 13 amino acids at the C-terminus of the ‘a’ isoform [6]. For human Them1 (Them1) and a truncated Them1 containing the two thioesterase domains, but lacking the START domain (Them1-ΔSTART), a synthetic gene encoding Them1b (the human ortholog of mouse Them1) was codon optimized to achieve maximal recombinant protein expression (Thermo Fisher; Waltham, MA) and subcloned into a pET19b bacterial expression vector (Novagen, EMD Biosciences; Madison, WI), which introduced an in-frame N-terminal His-tag. Sufficient quantities of recombinant Them1 required for the HTS were obtained with the assistance of the the University of Georgia Bioexpression and Fermentation Core Facility (Athens, GA). Cultures of E. coli Shuffle T7 competent cells (New England Biolabs, Ipswich, MA) transformed with pET19b-Them1 plasmid were grown to an A₆₀₀ of ∼0.5–0.7 in terrific broth followed by induction of recombinant Them1 by the addition of 0.5 mM isopropyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C. The bacteria were harvested by centrifugation and then lysed with 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 150 mM imidazole and 1 mM β-mercaptoethanol. The soluble fraction following centrifugation of the bacterial lysate was purified by fast protein liquid chromatography (FPLC) using a HisTrap affinity column (HisTrap HP column; GE Healthcare, Waukesha, WI) after equilibration with 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 150 mM imidazole and 1 mM β-mercaptoethanol, and then washed with the same buffer. Recombinant Them1 was eluted from the HisTrap HP column using 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 500 mM imidazole and 1 mM β-mercaptoethanol. To further increase purity, recombinant Them1 was applied to a HisTrap HP affinity column (HisTrap HP column; GE Healthcare, Waukesha, WI) and re-purified as described above followed by dialysis into buffer containing 20 mM Tris-Cl (pH 7.4), 500 mM NaCl and 10% glycerol using a slide-a-lyzer dialysis cassette (Thermo Fisher; Waltham, MA). Purity of recombinant Them1 was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. The concentration of recombinant Them1 was determined according to the molar extinction coefficient at 280 nm, which was calculated based on the amino acid sequence (www.expasy.org). Single use aliquots of recombinant Them1 were stored at −80 °C until use to prevent protein precipitation during purification. The pH values of all buffers were adjusted to remain at least two units removed from the pI of recombinant Them1.

Molecular cloning, and bacterial expression and purification of recombinant Acot isoforms were prepared as previously described with modifications [7]. The open reading frames of mouse Acot1 (Acot1), mouse Them1 and human Acot12 (Acot12) were amplified by PCR using the template plasmids ds-RED-Acot1, pCMV-SPORT6-Them1 and pcDNA-Acot12, respectively. The open reading frames of mouse Acot2 (Acot2) and human Acot13 (Acot13) were amplified by PCR using mouse liver tissue. The genes were subcloned into pET19b or pET29b bacterial expression vectors (Novagen, EMD Biosciences; Madison, WI), which introduced an in-frame N- or C-terminal His-tag, respectively. Maltose-binding protein (MBP)-tagged human Acot9 (Acot9) plasmid was prepared as previously described [10]. These plasmids were transformed into E. coli strains BL21 (DE3) (New England Biolabs; Ipswich, MA) or Shuffle T7 competent cells (New England Biolabs, Ipswich, MA), grown to an A₆₀₀ of ∼0.5–0.7 in terrific broth and then induced by the addition of 0.5 mM isoproyl β-D-thiogalactoside with 16 h of shaking (250 rpm) at 18 °C. The bacteria were harvested by centrifugation at 10,000 ×g for 20 min at 4 °C, and then lysed with 20 mM Tris-Cl, (pH 7.4) 500 mM NaCl, 150 mM imidazole, 1 mM β-mercaptoethanol and ethylenediaminetetraacetic acid-free protease inhibitors (Sigma Aldrich, St. Louis, MO). Following centrifugation of the bacterial lysate at 20,000 ×g for 20 min at 4 °C, recombinant Acot isoforms were purified by FPLC using a HisTrap affinity column (HisTrap HP column; GE Healthcare, Waukesha, WI) unless otherwise specified. The soluble fractions containing recombinant Acot isoforms were applied to the column after equilibration with 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 150 mM imidazole, 1 mM β-mercaptoethanol and ethylenediaminetetraacetic acid-free protease inhibitors (Sigma Aldrich, St. Louis, MO), and then washed with the same buffer. His-tagged recombinant Acot isoforms were then eluted from the HisTrap HP column using 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 500 mM imidazole, 1 mM β-mercaptoethanol and ethylenediaminetetraacetic acid-free protease inhibitors (Sigma Aldrich, St. Louis, MO). The soluble fraction containing MBP-tagged recombinant Acot9 was applied to a MBPTrap HP column after equilibration with 20 mM Tris-Cl, (pH 7.4) 200 mM NaCl, 1 mM EDTA and protease inhibitors (Sigma Aldrich, St. Louis, MO), washed with the same buffer and then eluted with 20 mM Tris-Cl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM maltose and protease inhibitors (Sigma Aldrich, St. Louis, MO). Following purification, all recombinant Acot isoforms were subjected to dialysis into buffer containing 20 mM Tris-Cl (pH 7.4), 500 mM NaCl and 10% glycerol using a slide-a-lyzer dialysis cassette (Thermo Fisher; Waltham, MA). Assessment of purity, protein concentration and storage of single use aliquots of recombinant Acot isoforms, and adjustment of buffer pH values were performed as described above for recombinant Them1.

Recombinant human Them1 START domain (START; amino acids 339–594 of Them1b) was prepared as previously described [8]. A synthetic gene encoding the START domain was cloned into a pNIC28-Bsa4 bacterial expression vector (Novagen, EMD Biosciences; Madison, WI), which introduced an in-frame N-terminal His₆ fusion containing a tobacco etch virus protease cleavage site to facilitate tag removal. The pNIC28-Bsa4-START domain plasmid was co-transformed into E. coli BL21 (DE3) (New England Biolabs; Ipswich, MA) competent cells with a pG-Tf2 vector (encoding groES-gorEL-tig chaperones) and then grown to an A₆₀₀ of ∼0.5–0.7 in terrific broth. Under conditions of shaking (250 rpm) at 18 °C, chaperones were induced by the addition of 5 ng/mL tetracycline HCl for 60 min followed by START domain induction upon the addition of 0.5 mM isoproyl β-D-thiogalactoside for ∼ 18 h. The bacteria were harvested by centrifugation, and then lysed by sonication with 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 25 mM imidazole, 5% glycerol, lysozyme, Dnase A, 0.1% Triton X-100, 5 mM β-mercaptoethanol and 100 μM phenylmethylsulfonyl fluoride. Following centrifugation of the bacterial lysate, recombinant START was purified by FPLC using a His affinity column (HisTrap HP column; GE Healthcare; Waukesha, WI). The soluble fraction containing recombinant START was applied to the column after equilibration with 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 25 mM imidazole and 5% glycerol, and then washed with the same buffer. Recombinant START was eluted from the HisTrap HP column using 20 mM Tris-Cl (pH 7.4), 500 mM NaCl, 500 mM imidazole and 5% glycerol followed by tobacco etch virus protease-mediated His-tag cleavage at 4 °C overnight with simultaneous dialysis into 20 mM Tris-Cl (pH 7.4), 500 mM NaCl and 5% glycerol. Recombinant START was further purified using a Superdex column (HiLoad 16/60 Superdex 75 column; GE Healthcare; Waukesha, WI) in 20 mM Tris-Cl (pH 7.4), 500 mM NaCl and 5% glycerol. Assessment of purity, protein concentration and storage of single use aliquots of recombinant START were performed as described above for recombinant Them1. Recombinant His-tag human (PC-TP, synonym: StARD2) was expressed and purified as previously described [11].