Department of Chemical and Biological Engineering, Korea University, 136-713 Anam-Dong 5-1, Seoul, Republic Korea.
Biotechnol Bioeng. 2012 Feb;109(2):325-35. doi: 10.1002/bit.23320. Epub 2011 Sep 9.
As a fusion partner to express aggregation-prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N- and C-domain) and reportedly has a high structural stability. When the full-length ePGK (F-ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N-domain of ePGK (N-ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N-ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self-assembly function, L-arginine-degrading activity, and the correct secondary structure, respectively, through the fusion expression using N-ePGK. These results indicate that N-ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli.
作为表达聚集倾向的异源蛋白的融合伴侣，我们研究了由两个功能域（N 域和 C 域）组成的大肠杆菌磷酸甘油酸激酶（ePGK），据报道它具有很高的结构稳定性。当全长 ePGK（F-ePGK）用作融合伴侣时，异源蛋白的可溶性增加，但其中一些仍有很大比例的不溶性聚集体。令人惊讶的是，使用 ePGK 的 N 域（N-ePGK）进行融合表达使所有测试的异源融合蛋白的不溶性部分显著减少到 10%以下。此外，我们评估了 N-ePGK 在使靶蛋白以其自身天然功能或结构表达的效果。结果发现，人铁蛋白轻链、细菌精氨酸脱亚氨酶、人粒细胞集落刺激因子通过使用 N-ePGK 进行融合表达，分别具有自我组装功能、L-精氨酸降解活性和正确的二级结构。这些结果表明，N-ePGK 是一种高效的融合伴侣，可广泛用于在大肠杆菌中合成各种异源蛋白。
