Comparison of Various Methods for Periplasmic Release of Recombinant Creatinase from Escherichia coil

以不同方法自大腸桿菌細胞間質釋放重組蛋白之比較

陳昱丞(Yu-Cheng Chen) ； 陳樹人(Shu-Jen Chen) ； 張敏政(Ming-Chung Chang) ； 陳特良(Teh-Liang Chen)

《Journal of the Chinese Institute of Chemical Engineers》 36卷5期 (2005/09) Pp. 527-532

https://doi.org/10.6967/JCICE.200509.0527

摘要

本文比較以不同方法自大腸桿菌細胞間質釋放重組肌酸酵素。所討論的方法包括細胞破碎（超音波和甲苯／乙醇）、化學通透（Triton X-100、胍鹽酸、胍鹽酸／EDTA、氯仿、甘胺酸）、和滲透壓衝擊。實驗發現，牽涉到去除脂多醣的方法（如胍鹽酸／EDTA和滲透壓衝擊），可以同時得到高回收率和高酵素純度。此結果顯示，脂多醣之不存在與間質釋放的通透性有密切的關係。相較於滲透壓衝擊，胍鹽酸／EDTA的間質釋放速率甚慢，這是因為前者是利用細胞收縮－膨脹的作用將間質蛋白送出，而後者僅是藉由擴散。滲透壓衝擊的效率可以利用溶菌素或Ca(上標 2+)前處理增進之。包含Ca(上標 2+)前處理的滲透壓衝擊，似乎是間質釋放的最適合方法。

並列摘要

Various methods are compared for the periplasmic release of a recombinant creatinase from Escherichia coli. The examined methods include cell disruption (sonication and toluene/ethanol), chemical permeabilization (Triton X-100, guanidine-HCl, guanidine-HCl/EDTA, chloroform, and glycine), and osmotic shock. It is found that methods involving the removal of lipopolysaccharide (such as guanidine-HCl/EDTA and osmotic shock) can achieve both excellent recovery and high purity. These findings indicate that the absence of lipopolysaccharide is closely related to the permeability for periplasmic release. Since it is based on diffusion, guanidine-HCl/EDTA achieves a much slower rate of periplasmic release than osmotic shock, which is based on shrinkage-and-swelling pumping. Osmotic shock can be further improved by using lysozyme or Ca(superscript 2+)-pretreatment, where the latter is seemingly the most suitable method for periplasmic release.

並列關鍵字

Periplasmic release ； Osmotic shock ； Escherichia coli ； Creatinase