Laboratory of Biotransformation and Biocatalysis, School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou, Henan, 450000, People's Republic of China; School of Food and Bioengineering/Collaborative Innovation Center for Production and Safety, Zhengzhou University of Light Industry, Zhengzhou, Henan, 450001, People's Republic of China.
Laboratory of Biotransformation and Biocatalysis, School of Food and Biological Engineering, Zhengzhou University of Light Industry, Zhengzhou, Henan, 450000, People's Republic of China; School of Food and Bioengineering/Collaborative Innovation Center for Production and Safety, Zhengzhou University of Light Industry, Zhengzhou, Henan, 450001, People's Republic of China.
Protein Expr Purif. 2021 Feb;178:105777. doi: 10.1016/j.pep.2020.105777. Epub 2020 Oct 15.
Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.
吡咯喹啉醌 (PQQ) 已被确认为除了众所周知的烟酰胺 (NAD(P)) 和黄素 (FAD、FMN) 之外的第三类氧化还原辅因子。它在各种生物体中发挥着重要的生理作用，具有很强的抗氧化性能。PQQ 的生物合成途径涉及一个由 4-7 个基因组成的基因簇，称为 pqqA-G，其中 pqqA 是 PQQ 合成的关键基因，编码 PqqA 前体肽。为了在大肠杆菌中生产重组 PqqA，融合标签被用于提高肽的稳定性和溶解性，并简化发酵过程的放大。在本文中，来自氧化葡萄糖酸杆菌 621H 的 pqqA 作为 SUMO 融合蛋白在大肠杆菌 BL21 (DE3) 中表达，并使用六组氨酸 (His6) 标签进行纯化。SUMO 融合蛋白和 His6 标签被 SUMO 特异性 ULP 蛋白酶特异性识别和切割，并使用固定化金属亲和层析获得高纯度的前体肽 PqqA。通过 Tricine-SDS-PAGE 确认目标蛋白的表达和纯化。最后，通过 LC-MS 确认了体外无细胞酶反应中 PQQ 的合成。
