Kouyoumdjian A J M, Lazar R A, Slater N K H
Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom.
Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA, United Kingdom.
J Chromatogr A. 2016 Oct 14;1468:64-72. doi: 10.1016/j.chroma.2016.09.006. Epub 2016 Sep 3.
In this study, the surface of a microporous walled micro-capillary film (MMCF) was modified into a weak anion exchanger by coupling cyanuric chloride and 2-diethylaminoethylamine (DEAE) to the ethylene-vinyl alcohol (EVOH) matrix. Fourier transform infrared spectroscopy (FTIR) measurements of modified and unmodified MMCFs confirmed the addition of a triazine ring and DEAE onto the membrane. Binding of bovine serum albumin (BSA) at pH 7.2 was found to follow a Langmuir isotherm with a maximum equilibrium binding of 12.4mg BSA/mL adsorbent and 8.2mg BSA/mL adsorbent under static and flow conditions, respectively. The ion exchange capacity, determined by Mohr's titration of chlorine atoms displaced from the functionalised surface, was found to be 195±21μmol Cl/mL of adsorber, comparable to commercial ion exchangers. BSA adsorption onto the ion exchanger was strongly pH-dependant, with an observed reduction in binding above pH 8.2. Frontal experiments of a BSA (5mg/mL) and lysozyme (5mg/mL) mixture demonstrated successful separation of BSA from lysozyme at more than 97% purity as verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separation between similarly charged anionic molecules was also achieved using BSA (5mg/mL) and herring sperm DNA (0.25mg/mL). BSA was extracted at 100% purity, demonstrating the ability of MMCF-DEAE to remove significant DNA contamination from a protein solution. These experiments highlight the potential for MMCFs to be used for fast protein purification in preparative chromatography application.
在本研究中，通过将三聚氯氰和2-二乙氨基乙胺（DEAE）偶联到乙烯-乙烯醇（EVOH）基质上，将微孔壁微毛细管膜（MMCF）的表面改性为弱阴离子交换剂。对改性和未改性的MMCF进行傅里叶变换红外光谱（FTIR）测量，证实了膜上添加了三嗪环和DEAE。发现在pH 7.2条件下，牛血清白蛋白（BSA）的结合遵循朗缪尔等温线，在静态和流动条件下，最大平衡结合量分别为12.4mg BSA/ mL吸附剂和8.2mg BSA/ mL吸附剂。通过莫尔滴定法测定从功能化表面置换出的氯原子，发现离子交换容量为195±21μmol Cl/ mL吸附剂，与商业离子交换剂相当。BSA在离子交换剂上的吸附强烈依赖于pH值，在pH 8.2以上观察到结合量减少。对BSA（5mg/mL）和溶菌酶（5mg/mL）混合物进行的前沿实验表明，经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）验证，成功地从溶菌酶中分离出了纯度超过97%的BSA。使用BSA（5mg/mL）和鲱鱼精DNA（0.25mg/mL）也实现了带相同电荷的阴离子分子之间的分离。以100%的纯度提取了BSA，证明了MMCF-DEAE从蛋白质溶液中去除大量DNA污染的能力。这些实验突出了MMCF在制备色谱应用中用于快速蛋白质纯化的潜力。
