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首页> 外文期刊>Journal of Applied Polymer Science >Thermosensitive poly(N-isopropylacrylamide) hydrogel for refolding of recombinant bovine prethrombin-2 from E-coli inclusion bodies
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Thermosensitive poly(N-isopropylacrylamide) hydrogel for refolding of recombinant bovine prethrombin-2 from E-coli inclusion bodies

机译：用于从大肠杆菌包涵体重新折叠重组牛凝血酶原2的热敏聚（N-异丙基丙烯酰胺）水凝胶

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The poly(N-isopropylacrylamide) (PNIPA) hydrogel, which is a kind of temperature-sensitive polymer, was synthesized by inverse suspension polymerization. The microscopy and scan electron microscopy (SEM) of PNIPA hydrogel were studied. The microscope photograph showed that the particles were in the range of 0.2-0.5mm in diameter, with numerous conjoint pores about 1-2 mu m spreading all over the surface of the beads. The swelling properties of PNIPA gel beads indicated that the tower critical solution temperature (LCST) of the gel was 33 degrees C. The PNIPA prepared was applied to the renaturation of bovine prethrombin-2 (pThr-2) from inclusion bodies produced in E. coli. It was observed that PNIPA was quite efficient in assisting protein renaturation at high protein concentration. When mixing with 105mg/mL PNIPA hydrogel during the refolding, the total activity of the thrombin was about 6222U/mL, compared with only 2800U/mL by simple dilution refolding. The kinetics of pThr-2 refolding with the absence or the presence of PNIPA was also studied respectively. The time required for the refolding with PNIPA gel was a little bit longer than that by the dilution method owing to the diffusion resistance of the protein into the network of the gel and the hydrophobic interaction between the protein and the polymer. The mechanism of the enhancement for the PNIPA gel to the refolding was further discussed. The porosity of the PNIPA hydrogel allows penetration of the unfolded protein into the inside of the polymer with a hydrophobic side chain, which can facilitate the formation of intermediate via hydrophobic interaction with the unfolded protein and the folding intermediate that are liable to re-aggregation. About 1.2mg of purified active thrombin could be recovered from I L of cells, which greatly facilitated the scale-up to the quantities of protein necessary for further functional and structural studies. A novel protein renaturation method mediated by PNIPA hydrogel beads, which highly increases the refolding efficiency with easy handling, recycling, and low cost, was proposed. (c) 2005 Wiley Periodicals, Inc.
机译：通过逆悬浮聚合法合成了一种对温度敏感的聚合物聚（N-异丙基丙烯酰胺）（PNIPA）水凝胶。研究了PNIPA水凝胶的显微镜和扫描电子显微镜（SEM）。显微镜照片显示该颗粒的直径在0.2-0.5mm范围内，在珠的整个表面上散布着约1-2μm的许多结合孔。 PNIPA凝胶珠的溶胀性能表明凝胶的塔临界溶液温度（LCST）为33摄氏度。制备的PNIPA用于从E中产生的包涵体对牛凝血酶原2（pThr-2）进行复性。大肠杆菌。观察到PNIPA在高蛋白质浓度下非常有效地协助蛋白质复性。当在重折叠过程中与105mg / mL PNIPA水凝胶混合时，凝血酶的总活性约为6222U / mL，而通过简单的稀释重折叠仅为2800U / mL。还分别研究了在不存在或不存在PNIPA的情况下pThr-2重折叠的动力学。由于蛋白质在凝胶网络中的扩散阻力以及蛋白质与聚合物之间的疏水相互作用，因此用PNIPA凝胶重折叠所需的时间比稀释方法要长一些。进一步讨论了PNIPA凝胶增强重新折叠的机制。 PNIPA水凝胶的孔隙率允许未折叠的蛋白质通过疏水侧链渗透到聚合物内部，这可以通过与未折叠的蛋白质和易于折叠的折叠中间体的疏水相互作用，促进中间体的形成。从I L细胞中可以回收到约1.2mg的纯化的活性凝血酶，这极大地促进了规模扩大至进一步功能和结构研究所需的蛋白质数量。提出了一种由PNIPA水凝胶珠介导的新的蛋白质复性方法，该方法可大大提高复性效率，且易于操作，回收和低成本。 （c）2005年Wiley Periodicals，Inc.

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