Superdex 200 prep grade

The raw skim milk was subjected to size exclusion chromatography to separate casein micelles using a gel filtration column (Superdex 200 prep grade, GE Life Sciences, Piscataway, NJ) connected to an AKTA fast-performance liquid chromatography unit (GE Life Sciences) using a previously reported method (Cheema et al., 2015) . Briefly, protein-free serum from the same animal was used as the mobile phase in size exclusion chromatography to ensure a native environment for the casein micelles. The protein-free serum was prepared by tangential flow cross-filtration of skim milk using a regenerated cellulose membrane with a 3-kDa molecular weight cut off (PLBC Prep scale TFF Cartridge, Millipore, Billerica, MA). The protein-free serum was preserved in 0.07% NaN 3 to prevent microbial growth and stored at 4°C until size exclusion chromatography. Two milliliters of raw skim milk was loaded onto the column and proteins were eluted using a flow rate of 1.0 mL/min for 2 column volumes (240 mL) and collected in 30-mL fractions. The elution of proteins was monitored using a UV absorbance detector set at 280 nm. A total of 4 replications were done for each sample. The casein micelle fractions were collected, freeze-dried, and stored at -20°C for the further analysis of the API by HPLC.

Bacterial growth and purification of Complex I and ATP synthase. The E. coli MWC215 (Sm R ndh::Cm R ) strain was grown in LB medium at 37°C in a 25 L fermenter and harvested at the late exponential growth phase. The membranes for Complex I purification were prepared by passing the cells through an APV Gaulin homogenizer. Then Complex I was purified by two consecutive chromatography steps using DEAE-Trisacryl M (Bio-Sepra) anion exchanger columns and gel filtration on Superdex 200 prep grade (GE Healthcare), respectively, as described 16 . ATP synthase of E. coli was expressed and purified as described previously 17
Reconstitution. Purified Complex I was reconstituted into liposomes as previously described 18 except that azolectin was suspended in the buffer containing MOPS-BTP 100 mM, pH 7.0, n-dodecyl β-D-maltopyranoside (DDM) 0.4%, Na-cholate 0.1%. A ratio of 1:10 (w/w), of the enzyme to azolectin was used unless other specified. ATP synthase and ATP synthase/Complex I coreconstitution were performed in the same way.
Preparation of subbacterial vesicles. The vesicles were prepared as described in 19 , but vesicles were loaded with 100 mM MOPS-BTP, 7.2, 50 mM KCl and 1 mM MgSO 4 , frozen and stored at -80°C until use.