The beads were rotated at 4��C for at least 6 h before pelleting at 1,300 g for 2 min. Beads were then washed three times with cold PBS, and GST fusion proteins were eluted using the same elution buffer as described for FPLC purification. GST-fusion proteins were further purified by FPLC using a 16 mm sellectchem �� 60 cm Superdex 75 gel filtration column (GE Healthcare) equilibrated with 50 mM NaH2PO4 and 150 mM NaCl (pH 7.2). The purified protein was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore, Billerica, MA). To cleave the GST tag, thrombin (1 unit/��l; GE Healthcare) was mixed with GST-fusion proteins (100 ��g/unit) and incubated at 23��C for at least 16 h. After 6 h rotation at 4��C, glutathione-coated beads (GE Healthcare) were added to pellet the GST tag.
Thrombin was used to cleave proteins while still bound to GST beads by incubation at 23��C for at least 16 h. Beads were then pelleted, leaving the untagged protein in the supernatant. Protein purities were assessed using SDS-PAGE gel and Coomassie Brilliant Blue staining. Protein concentrations were determined by using the molar absorption coefficient at 280 nm, which was calculated based on the primary sequence, or by using the Bradford method (8). Protein samples were used immediately or stored at ?80��C. Protein oligomerization Oligomerization was determined by FPLC using a Superose 6 10/300 GL column (GE Healthcare). The column was pre-equilibrated with the 50 mM NaH2PO4 and 150 mM NaCl (pH 7.2) before application of purified proteins (400 ��l of 5 ��M solutions).
Proteins were eluted with the same buffer at a flow rate of 0.5 ml/min at 23��C or 4��C. After collection in glass test tubes, fractions (300 ��l) were transferred to individual wells of a 96-well UV plate (Fisher Scientific). Relative protein concentrations were determined by A280 using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). The column was calibrated using a Gel Filtration Calibration Kit HMW (GE Healthcare), which allowed the determination of apparent molecular weights for recombinant proteins. To examine chemical effects on protein oligomerization, recombinant proteins were incubated with ATP (2 mM), ATP-��-S (0.5 mM), ADP (0.5 mM), palmitoyl-CoA (25 ��M), acetyl-CoA (50 ��M), CoASH (50 ��M), or myristic acid (25 ��M) for 30 min at 23��C or 37��C.
To insure its solubility at the concentrations used in this and subsequent experiments (11), myristic acid was first converted to its potassium salt using methanolic KOH and then dried under a stream of nitrogen Drug_discovery before being dissolved in aqueous buffer. Before FPLC, samples were ultrafiltered using a 10 kDa MWCO Microcon Centrifugal Filter Devices (Millipore) at 11,000 g for 40 min. Acyl-CoA thioesterase activity assay The acyl-CoA thioesterase activity of purified recombinant protein or tissue extracts was determined as previously described (8).
Related posts: