高效可溶性重组蛋白表达载体的构建

Construction of expression vectors for efficient expression of soluble recombinant proteins

本研究构建了两种高效表达可溶性重组蛋白的原核表达载体。一种载体由HisSUMO序列与pET30a(+)载体连接而成(命名为HisSUMO Express),表达的融合蛋白用Ni-NTA纯化,用SUMO蛋白酶I切割后可获得不留任何残基的重组蛋白。SUMO-蛋白酶I价格较贵,为减少表达蛋白的成本,第二种载体即在His-SUMO和目的序列之间加入羟胺切割位点(命名为HisSUMO Economic)。在HisSUMO Economic中表达的融合蛋白用Ni-NTA纯化,羟胺液切割后可获得仅留一个甘氨酸残基的重组蛋白。以在常规表达载体中难以表达的鼠源成纤维细胞生长因子-21(mFGF-21)为例,经葡萄糖消耗实验检测其活性,验证两种表达载体的效果。结果表明mFGF-21在两种载体中均获得了高效表达,融合蛋白占菌体总蛋白的40%以上,Ni-NTA纯化后的融合蛋白分别利用羟胺切割液和SUMO蛋白酶I切割,纯化的mFGF-21成熟蛋白回收量约为54mg/L,回收率约为6%。经两种载体表达后的mFGF-21蛋白均具有生物学活性,可促进脂肪细胞消耗葡萄糖,为进一步研究提供了基础。

The aim of the study is to construct two vectors for efficient expression of soluble recombinant proteins. The first vector was constructed by cloning the HisSUMO fragment into an expression vector pET30a（＋） to fuse with the gene of interest （designated as HisSUMO Express）. The second vector was constructed in the same way, but with a hydroxylamine cleavage site between HisSUMO and the gene of interest for an economic purpose （designated as HisSUMO Economic）. The mouse fibroblast growth factor-21（mFGF-21）, which was difficult to express in routine-used expression vectors, was taken as an example to test the vectors. The results showed that the mFGF-21 was expressed at high level in both vectors. The Sumo/mFGF-21 fusion protein accounted for more than 40% of the total bacterial protein. The fusion protein was purified with Ni-TNA column, and the HisSUMO was removed by cleavage of the fusion protein with either hydroxylamine solution or SUMO protease I. The concentration of the purified mFGF-21 mature protein was 54 mg/L and the recovery rate was 6%. The purified proteins derived from either hydroxylamine or SUMO protease I cleavage could stimulate glucose up-take by adipocytes. These results indicated that both HisSUMO Express and HisSUMO Economic were useful expression vectors for efficient expression of soluble recombinant proteins.