Effect of column dimensions and flow rates on size-exclusion refolding of beta -lactamase

We have investigated the effect of changing the column diam. and length on the size exclusion chromatog. (SEC) refolding of beta -lactamase from Escherichia coli-derived inclusion bodies (IBs). Inclusion bodies were recovered and solubilized in 6 M GdnHCl and 5 mM DTT. Up to 16 mg of denatured, solubilized beta -lactamase was loaded onto size exclusion columns packed with Sephacryl S-300 media (fractionation range: 104-1.5*106 Da). beta -Lactamase was refolded by eluting the loaded sample with 1 M urea in 0.05 M phosphate buffer, pH 7 at 23 DegC. The following columns were studied: 26*400, 16*400 and 26*200 mm, with a range of mobile phase flow rates from 0.33 to 4.00 mL/min. beta -Lactamase was successfully refolded in all three columns and at all flow rates studied. The beta -lactamase activity peak coincided with the major protein peak. Reducing the column diam. had little effect on refolding performance. The enzyme activity recovered was relatively independent of the mobile phase linear velocity. Reducing the column length gave a poorer resoln. of the protein peaks, but the enzyme activity peaks were well resolved. Calcn. of the partition coeffs. for beta -lactamase activity showed that the 26*400 column gave the greatest refolding performance. [on SciFinder (R)]