Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins
Aqueous-aqueous two-phase systems composed of low molecular weight of polyethylene glycols and dextrans for counter-current chromatographic purification of proteins
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DOI：
10.1016/j.jchromb.2006.07.028
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发表时间：
2006-12-05
影响因子：
3
通讯作者：
Ito, Yoichiro
中科院分区：
文献类型：
--
作者：
Shibusawa, Yoichi;Takeuchi, Naoko;Ito, Yoichiro
评价了由相对低分子量聚合物如聚乙二醇（PEG）（Mr：1000-1000）和葡聚糖（Mr：10，000和40，000）组成的新型水-水两相系统用于通过逆流色谱（CCC）纯化蛋白质。通过测定粘度和两相体积比等参数，优化了双水相体系的组成。用含有10 mM磷酸钾缓冲液（pH 9.0）的7.5% PEG 3350-10%葡聚糖T40系统成功地证明了从变形链球菌（SM）细胞裂解物中CCC纯化葡萄糖基转移酶（GTF）。CCC纯化后，CCC级分中所含的PEG和葡聚糖都很容易通过超滤在短时间内去除。通过酶活性以及十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳分析含有GTF的分级柱内容物。酶活测定表明，CCC组分中酶的回收率在95%以上。(c)2006 Elsevier B. V.保留所有权利。
New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-1000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities. (c) 2006 Elsevier B.V. All rights reserved.