Presence and removal of a contaminating NADH oxidation activity in recombinant maltose-binding protein fusion proteins expressed in Escherichia coli
Presence and removal of a contaminating NADH oxidation activity in recombinant maltose-binding protein fusion proteins expressed in Escherichia coli
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DOI：
10.2144/0000113822
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发表时间：
2012-04-01
期刊：
影响因子：
2.7
通讯作者：
Zhu, Guan
中科院分区：
文献类型：
--
作者：
Guo, Fengguang;Zhu, Guan
我们观察到在大肠杆菌中表达并使用传统的基于直链淀粉树脂的亲和层析纯化的麦芽糖结合蛋白（MBP）融合蛋白中存在污染性NADH氧化活性。这种污染性的NADH氧化活性可以用至少四种不同的来自表达为MBP融合蛋白的微小隐孢子虫的酶（即，来自I型脂肪酸合酶的烯酰还原酶结构域、脂肪酰辅酶A结合蛋白、来自聚酮化合物合酶的酰基连接酶结构域和推定的硫酯酶），而不管它们的NADH依赖性。然而，当融合蛋白被工程化以含有His标签并使用基于Ni-NTA树脂的方案纯化时，不存在污染性NADH氧化活性。或者，对于仅含有MBP标签的蛋白质，可以通过在细菌均质化和第一次柱洗涤期间向柱缓冲液中加入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活性。去除人工活性在使用NADH作为辅因子的酶的研究中是非常有价值的，特别是当天然活性低或重组蛋白无活性时。
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.