Kim Seonghun, Lee Sun Bok
School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31, Hyoja-dong, Pohang 790-784, Republic of Korea.
Protein Expr Purif. 2008 Nov;62(1):116-9. doi: 10.1016/j.pep.2008.06.015. Epub 2008 Jul 10.
Expression of archaeal proteins in soluble form is of importance because archaeal proteins are usually produced as insoluble inclusion bodies in Escherichia coli. In this study, we investigated the use of soluble fusion tags to enhance the solubility of two archaeal proteins, d-gluconate dehydratase (GNAD) and 2-keto-3-deoxy-D-gluconate kinase (KDGK), key enzymes in the glycolytic pathway of the thermoacidophilic archaeon Sulfolobus solfataricus. These two proteins were produced as inclusion bodies in E. coli when polyhistidine was used as a fusion tag. To reduce inclusion body formation in E. coli, GNAD and KDGK were fused with three partners, thioredoxin (Trx), glutathione-S-transferase (GST), and N-utilization substance A (NusA). With the use of fusion-partners, the solubility of the archaeal proteins was remarkably enhanced, and the soluble fraction of the recombinant proteins was increased in this order: Trx>GST>NusA. Furthermore, In the case of recombinant KDGKs, the enzyme activity of the Trx-fused proteins was 200-fold higher than that of the polyhistidine-fusion protein. The strategy presented in this work may contribute to the production of other valuable proteins from hyperthermophilic archaea in E. coli.
古菌蛋白以可溶形式表达很重要，因为古菌蛋白在大肠杆菌中通常以不溶性包涵体的形式产生。在本研究中，我们研究了使用可溶性融合标签来提高两种古菌蛋白——d-葡萄糖酸脱水酶（GNAD）和2-酮-3-脱氧-D-葡萄糖酸激酶（KDGK）的溶解度，这两种蛋白是嗜热嗜酸古菌嗜热栖热菌糖酵解途径中的关键酶。当使用多聚组氨酸作为融合标签时，这两种蛋白在大肠杆菌中以包涵体形式产生。为了减少大肠杆菌中包涵体的形成，GNAD和KDGK与三种蛋白融合，即硫氧还蛋白（Trx）、谷胱甘肽-S-转移酶（GST）和氮利用物质A（NusA）。通过使用融合伙伴，古菌蛋白的溶解度显著提高，重组蛋白的可溶部分按以下顺序增加：Trx>GST>NusA。此外，对于重组KDGK，与硫氧还蛋白融合的蛋白的酶活性比多聚组氨酸融合蛋白高200倍。本研究提出的策略可能有助于在大肠杆菌中生产来自超嗜热古菌的其他有价值的蛋白。
