Asayama Wakiko, Sawada Shin-ichi, Taguchi Hideki, Akiyoshi Kazunari
Institute of Biomaterials & Bioengineering, Tokyo Medical & Dental University 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Int J Biol Macromol. 2008 Apr 1;42(3):241-6. doi: 10.1016/j.ijbiomac.2007.11.003. Epub 2007 Nov 23.
The chaperone-like activity of a nanogel of cholesteryl group-bearing pullulan (CHP) was compared with that of GroEL for refolding acid-denatured green fluorescent protein (GFP). The refolding of denatured GFP was carried out by dilution of acid-denatured GFP in the presence of the nanogel or GroEL. GFP fluorescence was increasingly repressed with increases in the concentration of the CHP nanogel or GroEL added to the dilution buffer. The concentrations of 50% inhibition of recovery of GFP fluorescence were 0.03 microM (GroEL) and 0.08 microM (CHP nanogel), respectively. The refolding was resumed by the addition of ATP into the GroEL (0.20 microM) system or by the addition of methyl-beta-cyclodextrin into the nanogel (0.20 microM) system. In the nanogel-GFP system, about 90% of the intensity was recovered within 10 min. The half time (t(1/2)) for refolding in the CHP nanogel system (36 s) is almost equal to that of the natural chaperone GroEL-GroES system.
将含胆固醇基团的支链淀粉纳米凝胶（CHP）的伴侣样活性与GroEL对酸变性绿色荧光蛋白（GFP）复性的活性进行了比较。变性GFP的复性是在纳米凝胶或GroEL存在下通过稀释酸变性GFP来进行的。添加到稀释缓冲液中的CHP纳米凝胶或GroEL浓度增加时，GFP荧光被越来越多地抑制。抑制GFP荧光恢复50%的浓度分别为0.03微摩尔（GroEL）和0.08微摩尔（CHP纳米凝胶）。通过向GroEL（0.20微摩尔）系统中添加ATP或向纳米凝胶（0.20微摩尔）系统中添加甲基-β-环糊精可恢复复性。在纳米凝胶-GFP系统中，约90%的强度在10分钟内恢复。CHP纳米凝胶系统中复性的半衰期（t(1/2)）（36秒）几乎与天然伴侣GroEL-GroES系统的半衰期相等。
