Optimization of Elastin-Like Polypeptide Fusions for Expression and Purification of Recombinant Proteins in Plants
Optimization of Elastin-Like Polypeptide Fusions for Expression and Purification of Recombinant Proteins in Plants
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DOI：
10.1002/bit.22278
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发表时间：
2009-06-15
影响因子：
3.8
通讯作者：
Brandle, Jim E.
中科院分区：
文献类型：
--
作者：
Conley, Andrew J.;Joensuu, Jussi J.;Brandle, Jim E.
对用于医疗和工业用途的重组蛋白的需求正在迅速扩大，植物现在被认为是一种高效、廉价的生产手段。虽然重组蛋白在转基因植物中的积累可能很低，但我们先前已经证明，与弹性蛋白样多肽（ELP）标签的融合可以显著提高植物叶片中一系列不同重组蛋白的产量。ELP是具有重复五肽序列（VGVPG）（n）的生物聚合物，其对于生物分离是有价值的，充当重组蛋白的非色谱纯化的热响应标签。为了确定重组蛋白积累及其后续纯化的最佳ELP大小，将各种ELP标签与绿色荧光蛋白、白细胞介素-10、红细胞生成素和单链抗体片段融合，然后在烟草叶片中瞬时表达。我们的结果表明，具有30个五肽重复的ELP标签提供了在反向转换循环（ITC）纯化期间小ELP标签（n=5-40）对重组蛋白积累的积极作用和较大ELP标签（n=80-160）对重组蛋白回收的有益作用之间的最佳折衷。此外，相对于N-末端ELP融合，ELP融合标签的C-末端取向产生更高水平的靶蛋白。重要的是，ELP标签对促红细胞生成素的受体结合亲和力没有不利影响，证明了这些标签的惰性性质。ELP融合标签的使用提供了一种用于增强植物中重组蛋白的生产，同时有助于其纯化的方法。Biotechnol. Bioeng. 2009;103：562-573. (C)2009 Wiley Periodicals，Inc.
The demand for recombinant proteins for medical and industrial use is expanding rapidly and plants are now recognized as an efficient, inexpensive means of production. Although the accumulation of recombinant proteins in transgenic plants can be low, we have previously demonstrated that fusions with an elastin-like polypeptide (ELP) tag can significantly enhance the production yield of a range of different recombinant proteins in plant leaves. ELPs are biopolymers with a repeating pentapeptide sequence (VGVPG)(n) that are valuable for bioseparation, acting as thermally responsive tags for the non-chromatographic purification of recombinant proteins. To determine the optimal ELP size for the accumulation of recombinant proteins and their subsequent purification, various ELP tags were fused to green fluorescent protein, interleukin-10, erythropoietin and a single chain antibody fragment and then transiently expressed in tobacco leaves. Our results indicated that ELP tags with 30 pentapeptide repeats provided the best compromise between the positive effects of small ELP tags (n=5-40) on recombinant protein accumulation and the beneficial effects of larger ELP tags (n=80-160) on recombinant protein recovery during inverse transition cycling (ITC) purification. In addition, the C-terminal orientation of ELP fusion tags produced higher levels of target proteins, relative to N-terminal ELP fusions. Importantly, the ELP tags had no adverse effect on the receptor binding affinity of erythropoietin, demonstrating the inert nature of these tags. The use of ELP fusion tags provides an approach for enhancing the production of recombinant proteins in plants, while simultaneously assisting in their purification. Biotechnol. Bioeng. 2009;103: 562-573. (C) 2009 Wiley Periodicals, Inc.