Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins
Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins
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DOI：
10.1110/ps.45201
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发表时间：
2001-03-01
期刊：
影响因子：
8
通讯作者：
Waugh, DS
中科院分区：
文献类型：
--
作者：
Fox, JD;Kapust, RB;Waugh, DS
蛋白质通常与大肠杆菌麦芽糖结合蛋白（MBP）融合，以提高其产量并促进其纯化。此外，乘客蛋白的稳定性和溶解性通常可以通过将其融合到MBP来改善。在之前与其他两种高可溶性融合伴侣的比较中，MBP在促进一系列易于聚集的蛋白质的溶解度方面明显上级。为了解释这一观察结果，我们提出，MBP可以作为一个通用的分子伴侣在融合蛋白的背景下，通过结合到乘客蛋白的聚集倾向折叠中间体，并防止他们的自我协会。MBP中的配体结合裂缝由于其疏水性质而被认为是肽结合的可能位点。我们通过用谷氨酸系统地替换裂缝内和周围的疏水氨基酸侧链来测试这一假设。这些突变都不影响MBP在其未融合状态下的产率或溶解度。然后测试每种MBP在与三种过客蛋白融合时促进溶解度的能力：绿色荧光蛋白、p16和E6。麦芽糖结合裂缝内的突变（W62 E、A63 E、Y155 E、W230 E和W340 E）对融合蛋白的溶解度几乎没有影响或没有影响。相比之下，靠近裂缝一端的三个突变（W232E、Y242E和I317E）显著降低了相同融合蛋白的溶解度。对溶解度影响最深远的突变显示出降低MBP的整体稳定性。
Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6, Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.