Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins
Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins
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DOI：
10.1002/pro.157
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发表时间：
2009-07-01
期刊：
影响因子：
8
通讯作者：
Chilkoti, Ashutosh
中科院分区：
文献类型：
--
作者：
Christensen, Trine;Amiram, Miriam;Chilkoti, Ashutosh
我们之前开发了一种纯化重组蛋白的方法，称为逆转换循环 (ITC)，无需柱层析。 ITC 利用与目标蛋白质融合的类弹性蛋白多肽 (ELP) 的反溶解度相变。在 ITC 中，重组 ELP 融合蛋白循环经历其相变，从而将 ELP 融合蛋白与其他大肠杆菌污染物分离。在此，我们研究了融合蛋白中 ELP 的位置对四种重组 ELP 融合蛋白的表达水平和纯化蛋白产量的影响。将 ELP 放置在目标蛋白（蛋白-ELP）的 C 末端会导致四种 ELP 融合蛋白的表达水平更高，这也意味着纯化蛋白的产量更高。融合蛋白的位置也对其比活性有显着影响，因为对于四种蛋白质中的三种，ELP-蛋白质构建体的比活性低于蛋白质-ELP 构建体。我们的结果显示蛋白质-ELP 和 ELP-蛋白质融合构建体之间的 mRNA 水平没有差异。相反，我们对这些结果提出了两种可能的解释：首先，两个方向的融合蛋白之间的 mRNA 翻译效率可能不同，其次，较低的蛋白表达水平和较低的比活性与将 ELP 放置在融合蛋白的 N 末端增加了错误折叠的比例和活性较低的构象异构体的情况一致，与 ELP 存在于蛋白质 C 末端的融合蛋白相比，这些构象异构体也优先被降解。
We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin-like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C-terminus of the target protein (protein-ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP-protein constructs have a lower specific activity than protein-ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein-ELP and ELP-protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N-terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C-terminal end of the protein.