Yu H, Ma Q, Lin J, Sun Y F, Zheng F
Department of Cardiology, Guangdong General Hospital, Guangdong Provincial Cardiovascular Institute, Guangdong Academy of Medical Sciences, Guangzhou, China.
Genet Mol Res. 2013 Dec 6;12(4):6372-8. doi: 10.4238/2013.December.6.4.
Escherichia coli is the most widely used host for the production of recombinant proteins. However, most eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. The interactions between human FHL2 protein and many types of proteins, including structural proteins, kinases, and several classes of transcription factor, have been found to have important roles in a variety of fundamental processes, including arrhythmia, hypertrophy, atherosclerosis, and angiogenesis. To achieve high-level expression of soluble recombinant human FHL2 protein in E. coli, we have constructed a recombinant expression plasmid, pGEX-4T-1-FHL2, in which we merged FHL2 cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac inducible promoter. Using this plasmid, we have achieved high expression of soluble FHL2 as a GST fusion protein in E. coli BL21. We have used the engineered plasmid (pGEX-4T-1-FHL2) and the modified E. coli strain to overcome the problem of removing the GST moiety while expressing soluble FHL2. Our results show that: 1) the recombinant plasmid was successfully constructed. Sequencing results showed that FHL2 and GST are in the same reading frame; 2) at 23°C, soluble GST-FHL2 fusion protein was highly expressed after induction with 0.1 mM IPTG; and 3) GST-FHL2 can be detected by Western blotting using mouse monoclonal anti-GST antibody. Our data are the first to show that high yields of soluble FHL2 tagged with GST can be achieved in E.coli.
大肠杆菌是生产重组蛋白时使用最广泛的宿主。然而，大多数真核蛋白通常以不溶性、错误折叠的包涵体形式获得，需要进行溶解和重折叠。已发现人类FHL2蛋白与多种类型的蛋白（包括结构蛋白、激酶和几类转录因子）之间的相互作用在包括心律失常、肥大、动脉粥样硬化和血管生成在内的各种基本过程中发挥重要作用。为了在大肠杆菌中实现可溶性重组人FHL2蛋白的高水平表达，我们构建了一个重组表达质粒pGEX-4T-1-FHL2，其中我们将FHL2 cDNA与tac诱导型启动子下游的谷胱甘肽S-转移酶（GST）编码序列融合。使用该质粒，我们在大肠杆菌BL21中实现了可溶性FHL2作为GST融合蛋白的高表达。我们使用了工程化质粒（pGEX-4T-1-FHL2）和改良的大肠杆菌菌株来克服在表达可溶性FHL2时去除GST部分的问题。我们的结果表明：1）成功构建了重组质粒。测序结果表明FHL2和GST处于同一阅读框；2）在23°C下，用0.1 mM IPTG诱导后，可溶性GST-FHL2融合蛋白高表达；3）使用小鼠单克隆抗GST抗体通过蛋白质免疫印迹法可检测到GST-FHL2。我们的数据首次表明在大肠杆菌中可以实现高产率的带有GST标签的可溶性FHL2。
