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

采用逆流色谱技术，研究了聚乙二醇和葡聚糖等小分子聚合物组成的新型双水相体系对蛋白质的分离纯化。通过测量粘度和两相体积比等参数，优化了双水相体系的组成。用7.5%PEG3350-10%葡聚糖T40体系，10 mM磷酸二氢钾缓冲液，在pH 9.0时，成功地从变种链球菌(SM)细胞裂解液中分离纯化了葡萄糖转移酶(GTF)。用含10 mM磷酸二氢钾缓冲液的7.0%PEG3350-10%葡聚糖T40体系，在pH 9.0的条件下，成功地从大肠杆菌细胞裂解液中分离纯化了一种MBP-HDAC。经过CCC纯化后，CCC馏分中的两种聚合物都可以在短时间内通过超滤去除。收集的含有目的蛋白的组份进行了基于高效液相色谱法的体外分析和十二烷基硫酸钠聚丙烯酰胺凝胶电泳法的分析。在CCC分离过程中，从融合HDAC中消化了MBP标签，并通过一步操作纯化了天然HDAC，并保持了脱乙酰酶的活性。

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.