| Frozen cell pellets were resuspended in 10 ml 0.1 M Tris pH 8 containing a cocktail of protease inhibitors (complete mini, Roche) for 100 ml of bacterial culture. They were sonicated for 45 s at room temperature and centrifuged for 10 min at 12000 g. The inclusion bodies pellet was then resuspended with the same volume ratio in 0.1 M Tris pH 8, 6 M guanidine-HCl (Gu-HCl), 5 mM DTT and rotated for 1-2 h on a rotary mixer. The extract was centrifuged at 12000 g for 10 min and the supernatant was put in a dialysis cassette of MWCO 3500 (Pierce) and submitted to the refolding protocol of Tsumoto et al. [K. Tsumoto, K. Shinoki, H. Kondo, M. Uchikawa, T. Juji, I. Kumagai, Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent- application to a human single-chain Fv fragment, J. Immunol. Methods 1998; 219:119-129.]. The concentration of Gu-HCl in the dialysis bath ( 1 l of 0.1 M Tris pH 8, for a 10 ml cassette) was decreased in the following steps: 6 M ? 3 M ? 2 M ? 1 M ? 0.5 M ? 0 M. Step changes were applied overnight for 6 M , overday for 3 M, overnight for 2 M, and so on. Oxidized glutathione (250 µM) and reduced glutathione (500 µM) were added at the 1 M Gu-HCl step and readded with 0.4 M arginine at the 0.5 M Gu-HCl step. Precipitation occurred in the last step but was not due to PSC33. The refolded protein was then applied to a semipreparative (10 x 250 mm) C18 (10 µm 300 ?) reverse phase HPLC column (Vydac) operated at 3 ml/min and 40°C with a Waters 600 pump. Buffer A was 0.1% TFA in water, Buffer B was 100% CH3CN + 0.04% TFA. One min fractions were analyzed by MALDI-TOF, freeze-dried individually, weighed and analyzed by SDS-PAGE, circular dichroism and immunoassay. |