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首页> 外文期刊>Biotechnology Progress >Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin
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Solid-phase refolding of cyclodextrin glycosyltransferase adsorbed on cation-exchange resin

机译：阳离子交换树脂吸附环糊精糖基转移酶的固相重折叠

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Expression with a fusion partner is now a popular scheme to produce a protein of interest because it provides a generic tool for expression and purification. In our previous study, a strong polycationic tail has been harnessed for an efficient purification scheme. Here, the same polycation tail attached to a protein of interest is shown to hold versatility for a solid-phase refolding method that utilizes a charged adsorbent as a supporting material. Cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at the C-terminus (CGTK10ase) retains the ability to bind to a cation exchanger even in a urea-denatured state. When the denatured and adsorbed CGTK10ase is induced to refold, the bound CGTK10ase aggregates little even at a g/L range. The renatured CGTK10ase can also be simply recovered from the solid support by adding high concentration of NaCl. The CGTK10ase refolded on a solid support retains specific enzyme activity virtually identical to that of the native CGTK10ase. Several factors that are important in improving the refolding efficiency are explored. Experimental results indicate that nonspecific electrostatic interactions between the charge of the ion exchanger and the local charge of CGTase other than the polycationic tag should be reduced to obtain higher refolding yield. The solid-phase refolding method utilizing a strong polycationic tag resulted in a remarkable increase in the refolding performance. Taken together with the previous report in which a series of polycations were explored for efficient purification, expression of a target protein fused with a strong polycation provides a straightforward protein preparation scheme.
机译：与融合伴侣的表达现在是产生目标蛋白的流行方案，因为它提供了表达和纯化的通用工具。在我们之前的研究中，利用强聚阳离子尾部进行了有效的纯化。在此，与目标蛋白质连接的同一聚阳离子尾巴显示出对于利用带电吸附剂作为支撑材料的固相复折方法具有通用性。与C端的10个赖氨酸残基融合的环糊精糖基转移酶（CGTase）（CGTK10ase）即使在尿素变性的状态下也保留了与阳离子交换剂结合的能力。当诱导变性和吸附的CGTK10ase重折叠时，即使在g / L范围内，结合的CGTK10ase也很少聚集。通过添加高浓度的NaCl，也可以简单地从固相支持物中回收被变性的CGTK10ase。重折叠在固体支持物上的CGTK10ase保留了与天然CGTK10ase几乎相同的特异性酶活性。探索了对提高重折叠效率很重要的几个因素。实验结果表明，应减少离子交换剂电荷与CGTase除聚阳离子标签以外的局部电荷之间的非特异性静电相互作用，以获得更高的重折叠产量。利用强聚阳离子标签的固相重折叠方法导致重折叠性能显着提高。与以前的报告（其中探索了一系列的聚阳离子进行有效纯化）一起，与强聚阳离子融合的靶蛋白的表达提供了一种简单的蛋白制备方案。

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