Activation of phosphatidylinositol transfer protein α and β isoforms from inclusion bodies

Fully active phosphatidylinositol transfer protein (PI-TP) isoforms alpha and beta have been obtained from Escherichia coli inclusion bodies. Folding and activation of PI-TP alpha was achieved in the presence of DiC7: 0-phosphatidylcholine-Triton X-114 (PtdCho-TX114) mixed micelles. Replacement of DiC7:0-PtdCho with the natural ligands of PI-TP alpha, i.e, long-chain PtdCho and phosphatidylinositol, did not stimulate activation. Efficient activation of PI-TP alpha required a low temperature (4 degreesC), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e. up to 500 mug ml(-1)). The inclusion bodies yielded 10 mg homogeneous PI-TP alpha per liter of E. coli culture. Conditions for full activation of PI-TP beta were similar to those for PI-TP alpha except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e. 10 mug ml(-1)) were required. In contrast to PI-TP alpha, PI-TP beta lost its lipid transfer activity within a few days. This inactivation could be prevented by addition of beta -alanine. In summary, despite 94% sequence similarity, PI-TP alpha and PI-TP beta display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding. (C) 2001 Elsevier Science B.V. All rights reserved.