Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
Biotechniques. 2012 Apr;52(4):247-53. doi: 10.2144/0000113822.
We observed the presence of contaminating NADH oxidation activity in maltose binding protein (MBP) fusion proteins expressed in Escherichia coli and purified using conventional amylose resin-based affinity chromatography. This contaminating NADH oxidation activity was detectable with at least four different enzymes from Cryptosporidium parvum expressed as MBP-fusion proteins (i.e., an enoyl-reductase domain from a type I fatty acid synthase, a fatty acyl-CoA binding protein, the acyl-ligase domain from a polyketide synthase, and a putative thioesterase), regardless of their NADH dependence. However, contaminating NADH oxidation activity was not present when fusion proteins were engineered to contain a His-tag and were purified using a Ni-NTA resin-based protocol. Alternatively, for proteins containing only an MBP-tag, the contaminating activity could be eliminated through the addition of 0.1% Triton X-100 and 2% glycerol to the column buffer during homogenization of bacteria and first column wash, followed by an additional wash and elution with regular column and elution buffers. Removal of the artifactual activity is very valuable in the study of enzymes using NADH as a cofactor, particularly when the native activity is low or the recombinant proteins are inactive.
我们观察到在大肠杆菌中表达并使用常规的淀粉亲和层析纯化的麦芽糖结合蛋白 (MBP) 融合蛋白中存在污染的 NADH 氧化活性。这种污染的 NADH 氧化活性可以检测到至少四种不同的来自微小隐孢子虫的 MBP 融合蛋白表达的酶（即，I 型脂肪酸合酶的烯酰还原酶结构域、脂肪酸酰基辅酶 A 结合蛋白、聚酮合酶的酰基连接酶结构域和假定的硫酯酶），而与它们是否依赖 NADH 无关。然而，当融合蛋白被设计为含有 His 标签并用基于 Ni-NTA 树脂的方案纯化时，就不会存在污染的 NADH 氧化活性。或者，对于仅含有 MBP 标签的蛋白质，可以通过在细菌匀浆和第一柱洗涤过程中向柱缓冲液中添加 0.1% Triton X-100 和 2%甘油，然后用常规柱和洗脱缓冲液进行额外的洗涤和洗脱来消除污染的活性。去除这种人为活性在使用 NADH 作为辅酶的酶研究中非常有价值，特别是当天然活性较低或重组蛋白无活性时。
