Franzmann Titus M
Department Chemie, Technische Universität München, 85747 Garching, Germany.
Int J Biol Macromol. 2006 Aug 15;39(1-3):104-10. doi: 10.1016/j.ijbiomac.2006.02.026. Epub 2006 Apr 19.
Recombinant gene expression in the prokaryotic host Escherichia coli is of general interest for both biotechnology and basic research. Use of E. coli is inexpensive and advantageous due to the fully developed genetic accessibility. However, often insoluble target protein (inclusion body) accumulates in the cell. Especially when producing eukaryotic or disulfide bridged proteins in E. coli, inclusion body formation is observed. Nonetheless, insoluble protein can be regained and refolded in vitro. Commonly, renaturation of proteins is accomplished by methods involving dilution and/or dialysis. An interesting alternative is matrix-assisted refolding in which the denatured protein is refolded in the immobilized state. Here, matrix-assisted refolding was applied to refold a double cysteine variant of Hsp26, a small heat-shock protein from Saccharomyces cerevisiae which was insoluble after biosynthesis in E. coli BL21 (DE3) cells. This oligomeric protein was efficiently recovered from the insoluble fraction and refolded to its native oligomeric and chaperone-active state using ion exchange and size exclusion chromatography.
原核宿主大肠杆菌中的重组基因表达在生物技术和基础研究中都具有普遍意义。由于其遗传可及性已充分发展，使用大肠杆菌成本低廉且具有优势。然而，目标蛋白往往会在细胞中积累形成不溶性物质（包涵体）。特别是在大肠杆菌中生产真核蛋白或含有二硫键的蛋白时，会观察到包涵体的形成。尽管如此，不溶性蛋白可以在体外重新获得并复性。通常，蛋白质的复性是通过稀释和/或透析等方法实现的。一种有趣的替代方法是基质辅助复性，即变性蛋白在固定状态下进行复性。在此，基质辅助复性被应用于复性酿酒酵母中的小分子热休克蛋白Hsp26的双半胱氨酸变体，该变体在大肠杆菌BL21(DE3)细胞中生物合成后不溶。使用离子交换色谱和尺寸排阻色谱，这种寡聚蛋白从不溶性部分中高效回收，并复性至其天然寡聚和伴侣活性状态。
