家蚕中一个新的膜蛋白基因BmMP16的融合表达与结晶初探
Expression of a new gene BmMP16 from Bombyx mori and the tentative crystallization of its purification
在本实验室构建的家蚕蛹 cDNA 文库中筛选到一条基因序列，经NCBI比对和跨膜预测分析后发现这是家蚕中一个新的家蚕膜蛋白基因，将其命名为BmMP16。将BmMP16基因与多角体基因ph融合后克隆到表达载体pGEX4T-1上，在Rosseta菌株中获得成功表达，且以包涵体的形式存在于沉淀中，其表达量约为45%。然后利用多角体蛋白能溶于碱性溶液的特性，将获得的沉淀超声后依次在8种不同pH的Tris-HCl缓冲液中溶解，再通过反复三次调节pH值和离心的方法获得纯化，蛋白纯度为73%，得率约为63%。这种纯化方法仅需要离心机与pH计就可以完成，是一种简便高效的膜蛋白纯化方法。然后对纯化得到的融合蛋白进行脱盐浓缩，通过结晶试剂盒对其结晶条件进行初步筛选，结果发现可能找到一种结晶条件，为进一步研究其功能打下基础。
We selected a gene named BmMP16 from the silkworm pupae cDNA library which is constructed by our laboratory. After blasting the sequence homology against the NCBI database, we identified it as an unknow membrane gene. According to the characterization of polyhedron, the BmMP16 gene was linked to the ph gene. The recombinant gene was expressed in E.coli system.The recombinant protein was expressed as inclusion bodies.We purify it by adjustted the pH of different buffers,then the recombinant protein became gradually soluble.We can obtain relatively pure protein by repeatedly changing the pH.The expression level of recombinant proteins is about 45%. Its purity reached at 73% and the yield was about 63%, so we find a new method.Then we concentrated and desalted the recombinant protein.We used the crystallization kit to select the condition.We possibily find the condition and this should lay a good basis for researching the structure of the menbrane protein BmMP16.
张烁烁、张耀洲、聂作明
分子生物学生物工程学昆虫学
家蚕膜蛋白多角体蛋白结晶
Bombyx morimenbrane proteinpolyhedrincrystallization
张烁烁,张耀洲,聂作明.家蚕中一个新的膜蛋白基因BmMP16的融合表达与结晶初探[EB/OL].(2011-05-13)[2025-08-03].http://www.paper.edu.cn/releasepaper/content/201105-306.点此复制
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