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首页> 外文期刊>Journal of structural and functional genomics >Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein
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Effect of N-terminal solubility enhancing fusion proteins on yield of purified target protein

机译：N端溶解度增强融合蛋白对纯化靶蛋白产量的影响

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We have studied the effect of solubilising N-terminal fusion proteins on the yield of target protein after removal of the fusion partner and subsequent purification using immobilised metal ion affinity chromatography. We compared the yield of 45 human proteins produced from four different expression vectors: three having an N-terminal solubilising fusion protein (the GB1-domain, thioredoxin, or glutathione S-transferase) followed by a protease cleavage site and a His tag, and one vector having only an N-terminal His tag. We have previously observed a positive effect on solubility for proteins produced as fusion proteins compared to proteins produced with only a His tag in Escherichia coli. We find this effect to be less pronounced when we compare the yields of purified target protein after removal of the solubilising fusion although large target-dependent variations are seen. On average, the GB1+His fusion gives significantly higher final yields of protein than the thioredoxin+His fusion or the His tag, whereas GST+His gives lower yields. We also note a strong correlation between solubility and target protein size, and a correlation between solubility and the presence of peptide fragments that are predicted to be natively disordered.
机译：我们已经研究了溶解N端融合蛋白对融合蛋白的去除和随后使用固定化金属离子亲和色谱法纯化后对目标蛋白产量的影响。我们比较了四种不同表达载体产生的45种人类蛋白质的产量：三种具有N末端可溶融合蛋白（GB1域，硫氧还蛋白或谷胱甘肽S-转移酶），其后是蛋白酶切割位点和His标签，以及一种仅具有N末端His标签的载体。以前我们已经观察到与仅在大肠杆菌中仅带有His标签的蛋白质相比，作为融合蛋白产生的蛋白质对溶解度具有积极影响。我们发现，当我们比较去除可溶性融合物后纯化的靶蛋白的产量时，虽然看到了大的靶标依赖性变异，但这种效果不太明显。平均而言，与硫氧还蛋白+ His融合物或His标签相比，GB1 + His融合物的蛋白质最终产量要高得多，而GST + His融合物的蛋白质最终产量要低得多。我们还注意到溶解度和目标蛋白质大小之间的强相关性，以及溶解度和被预测为天然无序的肽片段的存在之间的相关性。

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