Klermund Ludwig, Riederer Amelie, Groher Anna, Castiglione Kathrin
Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
Protein Expr Purif. 2015 Jul;111:36-41. doi: 10.1016/j.pep.2015.03.009. Epub 2015 Mar 21.
N-Acyl-d-glucosamine 2-epimerase (AGE) is an important enzyme for the biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac). Due to the wide range of biological applications of Neu5Ac and its derivatives, there has been great interest in its large-scale synthesis. Thus, suitable strategies for achieving high-level production of soluble AGE are needed. Several AGEs from various organisms have been recombinantly expressed in Escherichia coli. However, the soluble expression level was consistently low with an excessive formation of inclusion bodies. In this study, the effects of different solubility-enhancement tags, expression temperatures, chaperones and host strains on the soluble expression of the AGE from the freshwater cyanobacterium Anabaena variabilis ATCC 29413 (AvaAGE) were examined. The optimum combination of tag, expression temperature, co-expression of chaperones and host strain (His6-tag, 37°C, GroEL/GroES, E. coli BL21(DE3)) led to a 264-fold improvement of the volumetric epimerase activity, a measure of the soluble expression, compared to the starting conditions (His6-maltose-binding protein-tag, 20°C, without chaperones, E. coli BL21(DE3)). A maximum yield of 22.5mg isolated AvaAGE per liter shake flask culture was obtained.
N-酰基-D-葡萄糖胺2-差向异构酶（AGE）是生物催化合成N-乙酰神经氨酸（Neu5Ac）的一种重要酶。由于Neu5Ac及其衍生物具有广泛的生物学应用，其大规模合成备受关注。因此，需要合适的策略来实现可溶性AGE的高水平生产。来自各种生物体的几种AGE已在大肠杆菌中重组表达。然而，可溶性表达水平一直较低，且包涵体形成过多。在本研究中，考察了不同的溶解度增强标签、表达温度、伴侣蛋白和宿主菌株对来自淡水蓝藻多变鱼腥藻ATCC 29413的AGE（AvaAGE）可溶性表达的影响。与起始条件（His6-麦芽糖结合蛋白标签，20°C，无伴侣蛋白，大肠杆菌BL21(DE3)）相比，标签、表达温度、伴侣蛋白共表达和宿主菌株的最佳组合（His6标签，37°C，GroEL/GroES，大肠杆菌BL21(DE3)）使作为可溶性表达量度的体积差向异构酶活性提高了264倍。每升摇瓶培养物获得了22.5mg分离的AvaAGE的最大产量。
