Construction of wild-type human αA- and αB-crystallin cDNA expression clones has been previously described.
26,27 For all expression clones, plasmids were transformed into
E. coli strain BL 21(DE3) cells (Life Technologies). Seed cultures of 50 mL were started and grown overnight. Protein expression was performed in 4 × 400 mL cultures of M9CA plus trace metals and 100 μg/mL ampicillin as described previously.
26,27 Cultures were grown for 4 hours at 37°C to an OD
600 = ∼0.7. Cultures were induced with IPTG (final concentration of 1 mM) and grown overnight at 37°C. Bacteria were harvested by centrifugation at 5400
g for 15 minutes. The resulting pellet was suspended in 100 mL of N-lysis buffer (50 mM Tris 300 mM NaCl and 0.5 mM EDTA pH 7.5) and lysed by three passages in a French press (ThermoFisher, Waltham, MA) at 1500 psi. Lysed cells were centrifuged at 27,000
g for 30 minutes at 4°C. The soluble protein fraction was dialyzed overnight against 4 L of 5 mM sodium phosphate pH 7.5 and 0.5 mM DTT for TAT-αB or 50 mM Tris-HCl, 0.5 mM EDTA pH 7.4, and 0.5 mM DTT for other α crystallins (buffer A) in order to remove salts and optimize the buffer for ion exchange chromatography. Dialyzed protein was centrifuged again at 27,000
g for 30 minutes at 4°C. The supernatant material was then loaded onto an ion exchange column: hydroxyapatite (HA) for TAT-αB, Macro S for gC-αB, and Macro Q for wild type αA- or αB-crystallins. Following a column wash with either 100 mL of 100 mM sodium phosphate pH 7.5 and 0.5 mM DTT (buffer B: HA column) or 100 mL of buffer A (Macro Q/S columns), proteins were eluted with a 0 to 500 mM NaCl gradient in either buffer B or buffer A, respectively. Based on SDS-PAGE profiling, fractions positive for α-crystallin were pooled and concentrated using an Amicon pressure concentrator fitted with a 25-kDa molecular weight cutoff filter (Millipore, Billerica, MA). Recombinant α-crystallin proteins were further purified by gel-filtration using a column (Sephacryl S-400 HR; GE Healthcare Life Sciences, Pittsburgh, PA) and eluted with PBS. Fractions enriched for the protein of interest were pooled, concentrated as before, analyzed by 4% to 20% SDS-PAGE to confirm purity, and quantified using the BCA assay (Pierce, Rockford, IL). Purified protein was stored at 4°C, or at −80°C for long-term storage.