HIV-1整合酶的纯化及活性检测

Purification and Assays for Activities of HIV-1 Integrase

含整合酶基因的重组质粒 pET p31在E coliBL2 1(DE3)中以包含体形式高效表达HIV 1整合酶 (p31蛋白 )。初步纯化的包含体用 8mol/L尿素变性溶解 ,经阳离子交换剂柱层析和分子筛柱层析纯化 ,并通过梯度透析的复性过程去除尿素 ,获得可溶性及高纯度 p31。测定证实 ,复性后的 p31蛋白呈现良好的 3′加工。

A recombinant plasmid pET p31 containing an IN gene segment has been introduced into E coli BL21 (DE3) to express high level p31 in the form of inclusive bodies(IB) The wash purified p31 was dissolved in 8 mol/L urea and a simple purification method was developed by which could be obtained soluble and high purified p31 based on ion exchange chromatography, gel filtration and subsequent restrict renaturation Denatured and purified p31 was dialysed slowly against the buffer in which the urea concentration reduced gradually to remove the denaturation agent at last Most of the p31 molecules could be recovered and become soluble during the process The product showed significant activities of 3′ processing, chain transfer and disintegration