於逆流層析中以具有親和性反微胞萃取蛋白質溶菌酶

Abstract

逆流層析(countercurrent chromatography)是一種無固體靜相支持物之液相-液相分配層析法，常用於生物分子以及天然物的萃取以及分離。本實驗使用非離子型界面活性劑Span 85與藍色染料分子Cibacron Blue F-3GA(CB)結合，形成具有親和性的界面活性劑 CB-Span 85，由於CB染料分子具有與NAD+相似的結構，可與含核苷酸部位的蛋白質具有親和力，將CB-Span 85溶於有機溶劑形成反微胞相，可有效的提高反微胞的選擇率，可以只控制離子強度來調節蛋白質進出反微胞的效率。藉由CB-Span 85結合逆流層析來萃取三種蛋白質混合物(溶菌酶、牛清血蛋白、卵白蛋白)，在其反萃取時發現只有溶菌酶的存在，證明CB-Span 85對溶菌酶的親和力非常高。雖然溶菌酶之回收率會受到溶液中其他蛋白質之影響，本研究彰顯出此技術使用於蛋白質選擇性萃取之可行性。

Counter-current chromatography (CCC) is a technique of liquid-liquid chromatography which eliminates the use of solid support matrices. This method has been widely applied in extraction and separation of biomolecules and natural products. In this study, nonionic surfactant sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) to become an affinity surfactant (CB-Span 85) and to form affinity-based reversed micelles while dissolved in hexane. Since the CB molecule possesses a specific conformation that mimics nicotinamide adenine dinucleotide (NAD+), it can bind strongly and specifically to a wide range of nucleotide-dependent enzymes such as dehydrogenases and kinases. Moreover, high protein transfer efficiency in the reverse micelles can be easily achieved by controlling the ionic strength of the aqueous phase. Lysozyme was selectively extracted in a mixture of lysozyme, bovine serum albumin (BSA), and albumin using CB-Span 85 in CCC. This outcome has shown that the CB-Span 85 possesses high affinity interaction with lysozyme. Although lysozyme recovery was partially hindered by other proteins in the solution, this study demonstrates the feasibility of using the technique in selective protein extraction.