Refolding protein from inclusion bodies

The pellet obtained from the insoluble fraction was washed twice with 10 mM sodium phosphate buffer pH 6.0 containing 1% w/v Triton X‐100 (Fisher, electrophoresis grade), once with buffer containing 1 M sodium chloride and once with water. The inclusion bodies were dissolved by stirring at room temperature in 10 mL of denaturing buffer (7 M GuHCl, 50 mM sodium phosphate, pH 6.0, and 1 mM DTT for cysteine‐containing mutants, final protein concentration 1 mg/mL). Fast mixing of the protein into the denaturing buffer and dissolution of the inclusion bodies within a few minutes was found to be crucial to avoid aggregation of unfolded protein and low refolding yields. The solution of denatured protein was filtered through a 0.22 µm syringe filter (Millipore) or centrifuged (31,000g, 30 min) to remove any undissolved protein. Refolding by pulsed dilution was accomplished by the same protocol for native Bcx described above.