One-step purification of recombinant proteins by counter-current chromatography and analyses of their functions.

采用逆流色谱法（CCC）对低分子量聚合物（聚乙二醇（PEG）和葡聚糖）组成的新型水-水两相（AATP）体系纯化蛋白质进行了评价。通过测定两相间的粘度和体积比等参数，对两相体系的组成进行了优化。用7.5% PEG 3350-10%葡聚糖T40体系，含10 mM磷酸钾缓冲液，pH为9.0，成功地从突变链球菌（SM）细胞裂解液中纯化葡萄糖基转移酶（GTF）。AATP还用于麦芽糖结合蛋白标记组蛋白去乙酰化酶（MBP-HDAC）的CCC纯化。用7.0% PEG 3350-10%葡聚糖T40体系，含10 mM磷酸钾缓冲液，pH为9.0，成功地从大肠杆菌细胞裂解液中纯化了MBP-HDAC。CCC纯化后，CCC馏分中的两种聚合物均可在短时间内通过超滤去除。采用高效液相色谱法和十二烷基硫酸钠聚丙烯酰胺凝胶电泳对所收集的含有目标蛋白的组分进行分析。在CCC分离过程中，从融合HDAC中消化MBP标签，并通过一步操作纯化天然HDAC，保留了良好的脱乙酰酶活性。

New aqueous-aqueous two-phase (AATP) systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) and dextran 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 glucosyltransferase (GTF) from Streptococcus mutants (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.AATP were also evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by CCC. CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity.