Cloning, expression, and purification of LacY, YidC, SecYEG, and SecYEG-YidC

Engineering, expression, and purification of LacY were performed as described (40). Briefly, the C-terminal end of wild-type LacY was extended with a 36–amino acid–long unstructured polyGly polypeptide, followed by a His8-tag [GSM(G11)EAVEEAVEEA(G11)S(His8)] using QuikChange II polymerase chain reaction and plasmid pT7-5/LacY as a template. PolyGly LacY was purified from E. coli XL1-Blue (StrataGene) transformed with pT7-5 plasmids harboring given mutant genes by using Co(II) affinity chromatography as described. LacY eluted from the Co(II)-Talon column was concentrated and washed with 50 mM sodium phosphate (NaPi) (pH 7.5) and 0.01% (w/w) dodecyl-β-d-maltopyranoside (DDM; Maumee) on an Amicon Ultra-15 concentrator (EMD Millipore) with a 30-kDa cutoff.

Wild-type YidC with a His10-tag on the C terminus was cloned in pT7-7 plasmid and expressed and purified similar to LacY with a few exceptions: The expression strain used was E. coli BL21 (DE3), and the concentration of DDM was 0.03%. YidC eluted from the Co(II)-Talon column was concentrated and washed with 150 mM NaCl, 50 mM sodium phosphate (NaPi) (pH 7.5) and 0.03% (w/w) DDM on an Amicon Ultra-15 concentrator with a 30-kDa cutoff.

For cloning, expression and purification of SecYEG E. coli SF100 cells (41) bearing pTrc99a-SecYEG were grown in 2 liters of Luria-Bertani (LB) medium containing ampicillin (200 μg ml−1) and induced with 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) when the cultures reached an optical density at 600 nm of 0.8. After 2 hours of induction, the cells where harvested and shock-frozen in buffer 1 [20 mM tris-HCl (pH 8), 300 mM NaCl, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol (DTT)]. After cell disruption by a one-shot cell disruptor (Constant Systems LTD) and separating the cellular fragments by centrifugation, the membranes were harvested by ultracentrifugation and resuspended in buffer 1 (without DTT). For solubilization, 2% DDM was added and incubated for 2 hours at 4°C. After ultracentrifugation, the supernatant was incubated rotating with 0.5 ml of equilibrated Ni–nitrilotriacetic acid (NTA) and 30 mM imidazole in a total volume of 50 ml for 1 hour at 4°C. The Ni-NTA was separated from the buffer on a minicolumn and washed with 10-column bed volume (CV) washing buffer [20 mM tris-HCl (pH 8), 300 mM NaCl, 10% glycerol, 50 mM imidazole, and 0.05% DDM]. SecYEG was eluted with 10 CV elution buffer [20 mM tris-HCl (pH 8), 300 mM NaCl, 10% glycerol, 300 mM imidazole (pH 8), and 0.05% DDM]. The elution fractions were flash-frozen and stored at −80°C.

The purification of the SecY-YidC fusion construct (fig. S2) was identical to the SecYEG purification with some modifications: The expression strain for SecYEG-YidC fusion construct was Lemo21 (DE3). The cells were induced with 0.4 mM IPTG for 1 hour at 30°C. The pH of the buffers was 7.5, and for solubilization, 1% DDM was added. For immobilized metal affinity chromatography, 1 ml of equilibrated Ni-NTA and 20 mM imidazole in a total volume of 50 ml were used.

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