Sequential tentacle grafting and charge modification for enhancing charge density of mono-sized beads for facilitated protein refolding and purification from inclusion bodies
Sequential tentacle grafting and charge modification for enhancing charge density of mono-sized beads for facilitated protein refolding and purification from inclusion bodies
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连续触手嫁接和电荷修饰，用于增强单一尺寸珠子的电荷密度，以促进蛋白质重折叠和从包涵体中纯化
DOI：
10.1016/j.chroma.2014.04.055
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发表时间：
2014
影响因子：
4.1
通讯作者：
Yan Sun
中科院分区：
文献类型：
--
作者：
Xiao-Yan Dong;Ran Chen;Chun-Yan Yang;Yan Sun
我们之前已经发现，在复性溶液中加入相同电荷的介质可以通过抑制蛋白质的聚集来极大地提高纯蛋白质的复性。为此，制备了带负电荷的含磺酸基团的单一尺寸微球，以探讨同电介质对表达为包涵体(IBS)的增强型绿色荧光蛋白(EGFP)复性的促进作用。为了提高单体聚甲基丙烯酸缩水甘油酯(PgMA)微球(2.4gμm)的电荷密度，采用了一种连续的聚合物-触角接枝和磺酸盐修饰策略。也就是说，首先通过接枝聚合将GMA接枝到珠子上形成聚(GMA)触角，然后用亚硫酸钠对触须上的环氧基团进行改性，使其转化为磺酸基。通过这种方法制备的微球的电荷密度达到793kmol.moL/g，比未接枝的微球的电荷密度(285molmolg)提高了2.8倍左右。不同电荷密度的负电荷珠被用来促进IBS中同电荷态的EGFP的复性。随着电荷密度的增加，复性产率和复性速率增加。尿素微球和等电荷微球的抗聚集作用具有协同效应。此外，由于离子交换吸附使复性溶液中带正电的污染物蛋白质的去除率达到52%，因此实现了对EGFP的部分纯化。最后，通过重复的EGFP折叠和回收循环，证明了触手珠的可重用性。
We have previously found that addition of like-charged media in a refolding solution can greatly enhance the refolding of pure proteins by suppressing protein aggregation. Herein, negatively charged mono-sized microspheres with sulfonic groups were fabricated to explore the facilitating effect of like-charged media on the refolding of enhanced green fluorescent protein (EGFP) expressed as inclusion bodies (IBs). A sequential polymer-tentacle grafting and sulfonate modification strategy was developed to increase the charge density of mono-sized poly(glycidyl methacrylate) (pGMA) beads (2.4 μm). Namely, GMA was first grafted onto the beads by grafting polymerization to form poly(GMA) tentacles on the pGMA beads, and then the epoxy groups on the tentacles were converted into sulfonic groups by modification with sodium sulfite. By this fabrication strategy, the charge density of the beads reached 793 μmol/g, about 2.8 times higher than that modified without prior grafting of the pGMA beads (285 μmol/g). The negatively charged beads of different charge densities were used for facilitating the refolding of like-charged EGFP from IBs. The refolding yield as well as refolding rate increased with increasing charge density. The anti-aggregation effects of urea and like-charged microspheres were synergetic. In addition, partial purification of EGFP was achieved because the ion-exchange adsorption led to 52% removal of positively charged contaminant proteins in the refolded solution. Finally, reusability of the tentacle beads was demonstrated by repetitive EGFP refolding and recovery cycles.