Editing Refolding by Dialysis (section)

== On-column Refolding with beta-cyclodextrin ==

Inclusion body solubilization wascarried out in 8-M urea or 6-M guanidinium hydrochloride.Solubilized inclusion bodies were bound to Ni-NTA resin (Qi-agen, Valencia, California, USA) and pre-equilibrated in dena-turing buffer — by batch-absorption performed overnight atroom temperature. The amount of resin used is 5&nbsp;mg of protein/1 ml of resin. On-column renaturation and purificationwereperformed the next morning by several changes of buffers.First, the column was washed using the denaturing buffer con-taining 20 mM imidazole to remove nonspecifically-boundcontaminants. beta-mercaptoethanol at a final concentration of10 mM was added to this buffer and all following buffers if therefolding protein contained cysteines. 

In the next step, the column was washed with 10-column volumes (CV) of buffer A containing 0.1% Triton X-100 (buffer A (20 mM Tris-HCl, pH 8.0, 0.1 M NaCl). This was followed by a wash with 10 CV of Buffer A containing 5 mM beta-cyclodextrin (Sigma, St.Louis, Missouri, USA) to remove detergent from the pro-tein–detergent complex and to allow the protein to refold. Be-fore elution, an additional wash with 20 mM Tris-HCl, pH 8.0