Protein Purification
rTEV. The plasmid encoding rTEV protease was transformed intoE. coli BL21 Codon Plus and purified as described (Blommel &
Fox, 2007).
Acyl-CoA synthetases . Plasmids containing the coding sequences
for Sa MbcSWT,Sa MbcSK510A, and Rp IbuA, all fused to
cleavable N -terminal hexahistidine (His6) tags,
were overproduced and purified using a two-step procedure as described
(Crosby et al., 2012). Briefly, the plasmids were introduced intoE. coli BL21 C41λ (DE3) cells. The resulting strains were grown
in LB medium supplemented with 100 mg L-1 Ap at 37°C
first overnight in borosilicate tubes. The overnights were then
subcultured at a 1:20 ratio with shaking until early stationary phase
(OD600 ~2). A subculture was prepared
by diluting the initial culture at a 1:100 ratio into 500 mL of LB
medium supplemented with 100 mg L-1 Ap, and cells
were grown with shaking until reaching an OD600 of
0.6. Expression was induced using 0.5 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG), followed by overnight incubation
at 30°C. Cells were harvested by centrifugation at 8000 x g for 12
minutes, and the resulting pellets were stored at -80°C. Pellets were
resuspended in 30 mL of buffer 1 (50 mM Tris-HCl pH 8.0, 300 mM NaCl,
10 mM imidazole). Cells were lysed using a Digital Sonifier (Branson)
for 2- pulses separated by 5-s pauses on wet ice (25% amplitude) for
1.5-2 minutes. Cellular debris was removed by centrifugation at 38000
x g for 30 minutes at 4°C, and the soluble fraction was filtered using
a 0.45 μm filter. The protein was purified by two-step
Ni2+ affinity chromatography via a 4 mL bed volume
of nickel-nitrilotriacetic acid resin (Thermo Scientific)
pre-equilibrated with buffer 1 for 10 column volumes. The lysate was
loaded on the column, and the column was washed with wash buffer 2 (50
mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole) containing 1 mg
ml-1 lysozyme (Thermo Scientific) and 0.2mM
phenylmethanesulfonyl fluoride (PMSF) (Thermo Scientific). for a total
of seven column volumes. Proteins were eluted with buffer 3 (50 mM
Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole) over six column
volumes. Eluted protein fractions were analyzed using SDS-PAGE, and
those fractions containing the target protein were pooled. The
His6 tag was cleaved with rTEV protease using a 1:100
[v/v] rTEV to protein ratio overnight on at 4°C ice. Following the
cleavage, proteins were dialyzed sequentially against buffer 4 (50 mM
Tris-HCl pH 8.0, 300 mM NaCl, 0.5 mM EDTA), dialysis buffer 5 (50mM
Tris-HCl pH 8.0, 300 mM NaCl), and finally against buffer 1. The
proteins were reapplied to a pre-equilibrated
Ni2+-NTA column to capture the His6tag and rTEV. Untagged target proteins were isolated to apparent
homogeneity from the flow-through and dialyzed against buffer 6 (50 mM
Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EDTA, 20% [v/v] glycerol)
and then buffer 7 (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 20% glycerol).
Proteins were concentrated, drop-frozen in liquid nitrogen, and stored
at -80°C until used. Protein purity was evaluated using SDS-PAGE.