Tegel Hanna, Tourle Samuel, Ottosson Jenny, Persson Anja
School of Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Protein Expr Purif. 2010 Feb;69(2):159-67. doi: 10.1016/j.pep.2009.08.017. Epub 2009 Sep 4.
The effect of two Escherichiacoli expression strains on the production of recombinant human protein fragments was evaluated. High-throughput protein production projects, such as the Swedish Human Protein Atlas project, are dependent on high protein yield and purity. By changing strain from E. coli BL21(DE3) to E. coli Rosetta(DE3) the overall success rate of the protein production has increased dramatically. The Rosetta(DE3) strain compensates for a number of rare codons. Here, we describe how the protein expression of human gene fragments in E. coli strains BL21(DE3) and Rosetta(DE3) was evaluated in two stages. Initially a test set of 68 recombinant proteins that previously had been expressed in BL21(DE3) was retransformed and expressed in Rosetta(DE3). The test set generated very positive results with an improved expression yield and a significantly better purity of the protein product which prompted us to implement the Rosetta(DE3) strain in the high-throughput protein production. Except for analysis of protein yield and purity the sequences were also analyzed regarding number of rare codons and rare codon clusters. The content of rare codons showed to have a significant effect on the protein purity. Based on the results of this study the atlas project permanently changed expression strain to Rosetta(DE3).
评估了两种大肠杆菌表达菌株对重组人蛋白片段生产的影响。高通量蛋白质生产项目，如瑞典人类蛋白质图谱项目，依赖于高蛋白产量和纯度。通过将菌株从大肠杆菌BL21(DE3)更换为大肠杆菌Rosetta(DE3)，蛋白质生产的总体成功率显著提高。Rosetta(DE3)菌株可补偿一些稀有密码子。在此，我们描述了如何分两个阶段评估人基因片段在大肠杆菌菌株BL21(DE3)和Rosetta(DE3)中的蛋白质表达。最初，将一组先前在BL21(DE3)中表达的68种重组蛋白重新转化并在Rosetta(DE3)中表达。该测试集产生了非常积极的结果，蛋白产量提高，蛋白质产物的纯度显著提高，这促使我们在高通量蛋白质生产中采用Rosetta(DE3)菌株。除了分析蛋白质产量和纯度外，还对序列中的稀有密码子数量和稀有密码子簇进行了分析。稀有密码子的含量对蛋白质纯度有显著影响。基于本研究结果，图谱项目永久性地将表达菌株更换为Rosetta(DE3)。
