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

Abstract

We have investigated the effect of changing the column diameter and length on the size exclusion chromatography (SEC) refolding of β-lactamase from Escherichia coli-derived inclusion bodies (IBs). Inclusion bodies were recovered and solubilised in 6 M GdnHCl and 5 mM DTT. Up to 16 mg of denatured, solubilised β-lactamase was loaded onto size exclusion columns packed with Sephacryl S-300 media (fractionation range: 10 4–1.5×10 6 Da). β-Lactamase was refolded by eluting the loaded sample with 1 M urea in 0.05 M phosphate buffer, pH 7 at 23 °C. 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. β-Lactamase was successfully refolded in all three columns and at all flow rates studied. The β-lactamase activity peak coincided with the major protein peak. Reducing the column diameter 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 resolution of the protein peaks, but the enzyme activity peaks were well resolved. Calculation of the partition coefficients for β-lactamase activity showed that the 26×400 column gave the greatest refolding performance.