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

详细信息    查看全文

• 作者：Bouma ; Barend ; Westerman ; Jan ; Dekker ; Niek ; Gros ; Piet ; Wirtz ; Karel W.A.
• 关键词：Phosphatidylinositol transfer protein ; Phospholipid transport ; Inclusion body ; Mixed micelle ; Protein folding ; Phosphatidylcholine ; PI-TP ; phosphatidylinositol transfer protein ; C16 ; 0- ; C18 ; 2-PtdGro ; 1-palmitoyl- ; 2-linoleoyl-sn-phosphorylglycerol ; PyrPtd
• 刊名：Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular Enzymology
• 出版年：2001
• 出版时间：March 9, 2001
• 年：2001
• 卷：1546
• 期：1
• 页码：216-225
• 全文大小：252 K

文摘

Fully active phosphatidylinositol transfer protein (PI-TP) isoforms α and β have been obtained from Escherichia coli inclusion bodies. Folding and activation of PI-TPα 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-TPα, i.e. long-chain PtdCho and phosphatidylinositol, did not stimulate activation. Efficient activation of PI-TPα required a low temperature (4°C), the presence of dithiothreitol, and was achieved at a relatively high protein concentration (i.e. up to 500 μg ml⁻¹). The inclusion bodies yielded 10 mg homogeneous PI-TPα per liter of E. coli culture. Conditions for full activation of PI-TPβ were similar to those for PI-TPα except that long-chain PtdCho-TX114 mixed micelles and a very low protein concentration (i.e. 10 μg ml⁻¹) were required. In contrast to PI-TPα, PI-TPβ lost its lipid transfer activity within a few days. This inactivation could be prevented by addition of β-alanine. In summary, despite 94 % sequence similarity, PI-TPα and PI-TPβ display a striking difference both in their preference for the PtdCho acyl chain length required for activation, and in their conformational stability after folding.