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本实验利用重组ｒａｓ基因高效表达质粒ｐＪＬｃⅡｒａｓ在大肠杆菌ＴＡＰ１０６菌株中表达ｐ２１蛋白，该产物为融合蛋白，分子量在２３ｋＤ左右，具有ｒａｓ基因产物ｐ２１的全部生物学活性。在进行初步的酶切鉴定所构建质粒的组成后，本实验比较了４２℃水浴诱导不同时间菌体表达ｐ２１的效率，继而探讨了重组产物ｐ２１蛋白运用变性剂，通过变性──复性──层析的方法进行纯化的可行性，并检测了纯化产物的ＧＴＰ结合活性。结果表明，在４２℃水浴振荡诱导４ｈ可使ｐ２１表达量达到最大值，约占全部菌体蛋白总量的１５．６％；温度诱导表达的ｐ２１蛋白绝大部分以不溶状态存在于裂菌后沉淀中，经ＮＰ４０、脱氧胆酸钠等洗涤剂反复洗涤后，离心所得沉淀中ｐ２１ｒａｓ的纯度达８０％以上。将初步洗涤纯化的沉淀用８Ｍ尿素溶解，再通过缓慢的梯度透析逐步除去溶液中的尿素，在这一过程中相当一部分ｐ２１分子可逐渐复性为可溶解状态。利用阴离子交换柱层析可使复性后的ｐ２１蛋白得到进一步的纯化。在硝酸纤维素膜上进行的ＧＴＰ结合试验结果证明，经变性──复性──柱层析的纯化方法有效，纯化后的可溶性ｐ２１复性良好，ＧＴＰ结合活性明显提高。
In this study, p21 protein was expressed in Escherichia coli TAP106 strain by using recombinant plasmid pJLcⅡras, which is a fusion protein with a molecular weight of about 23 kD and possesses the full biological activity of ras gene product p21. After the initial restriction enzyme digestion of the constructed plasmid, we compared the efficiency of p21 expression in different temperature in 42 ℃ water bath, and then discussed the use of denatured p21 protein, Chromatography method to purify the feasibility and to detect the purified product GTP binding activity. The results showed that the expression of p21 reached the maximum after induced by water bath oscillation at 42 ℃ for 4 h, accounting for 15.6% of the total bacterial proteins. Temperature-induced expression of p21 protein existed mostly in the insoluble state Precipitate, the NP40, sodium deoxycholate and other detergents after repeated washing, the precipitate obtained by centrifugation p21ras purity of 80% or more. The initial wash purified precipitate was dissolved with 8M urea, and then gradually removed by slow gradient dialysis solution of urea, in the process a considerable part of p21 molecules can be gradually renatured to a soluble state. The renatured p21 protein was further purified by anion exchange chromatography. The results of GTP binding assay on nitrocellulose membrane demonstrated that the method of purification by denaturing-renaturation-column chromatography was effective, and the purified soluble p21 was refolded well and the GTP binding activity was significantly improved.