His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors
His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors
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DOI：
10.1023/b:jsfg.0000031965.37625.0e
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发表时间：
2004-01-01
期刊：
影响因子：
--
通讯作者：
Berglund, Helena
中科院分区：
文献类型：
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作者：
Woestenenk, Esmeralda A.;Hammarstrom, Martin;Berglund, Helena
我们比较了四种不同载体在大肠杆菌中表达带有N端或c端六组氨酸（His6）标签的蛋白质，并对20种人类蛋白质进行了测试。我们观察了每克干细胞重量的重组蛋白总产量水平，目标蛋白的溶解度，以及固定化金属离子亲和纯化纯化后的可溶性蛋白和总蛋白的产量。研究发现，一般情况下，N端和c端His6标签对蛋白质溶解度都有明显的负影响，但这种影响是靶蛋白特异性的。增溶性融合标签能够部分抵消这种负面影响。大多数靶蛋白可在变性条件下纯化，约有一半的蛋白可在生理条件下纯化。含c端His标记的构建体的蛋白产量和纯化蛋白产量最高。我们还观察到细胞生长速率的很大变化，我们确定这部分是由表达载体引起的，部分是由靶标引起的。发现这种变异与目标蛋白的生产水平、溶解度和三级结构含量无关。
We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative effect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with a C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.