本论文对人胰岛素原类似物的纯化方法进行了探讨。首先制备目标蛋白与钙调素的融合蛋白，并进行特异性蛋白酶切，后期实施三种技术对目标蛋白进行纯化，即镍柱亲和层析、等电点沉淀、热沉淀等，发现热沉淀能够去除大部分的杂蛋白，再进行镍柱亲和层析就可以得到纯度较高的目标蛋白分子，但是产率非常低。本论文又尝试构建目标蛋白的直接表达体系，目标蛋白成功表达。由于表达的蛋白是以包涵体的形式存在，所以利用尿素打开蛋白间的非共价键使其溶解，进一步通过亲和层析纯化目标蛋白，并经过后期复性和二硫键重组，得到较高生物学活性的人胰岛素原抗栓类似物。而且通过比较，发现直接表达方案蛋白的回收率有升高。

In this paper, two methods for the purification of human proinsulin analogues were discussed. First, the preparation of the fusion protein formed by target protein and calmodulin, and followed by specific protease digestion to produce target protein, the late implementation of the three techniques for purification of the target protein, including directly elution through Ni-column, isoelectric precipitation, and thermal precipitation. Found that heat can remove most of the impurities precipitated protein, and then nickel-affinity chromatography can obtain high purity target protein molecules, but the yield is very low. This paper has tried to build a direct target protein expression system, (to solve the above problems), the target protein was successfully expressed directly. Since protein expression in the form of inclusion bodies, so we use urea to resolve insoluble proteins and destroy the non-covalent bond, further purified target protein by affinity chromatography, and followed by refolding and disulfide reorganization, high antithrombotic activity of human proinsulin analogues were obtained.