Aqueous two-phase system formed by thermoreactive vinyl imidazole vinyl caprolactam copolymer and dextran for partitioning of a protein with a polyhistidine tail

A new type of aqueous two-phase system (ATPS) has been developed for application combining two attractive concepts in downstream processing: the immobilised metal affinity partitioning and the use of thermoprecipitating polymers. ATPS consisting of the thermoprecipitating copolymer of N-vinyl caprolactam/1-vinyl imidazole loaded with Cu ions (Cu-poly-VI-VCL) in the top phase and dextran T70 in the bottom phase was used for purification of recombinant lactate dehydrogenase carrying an affinity tag of 6 histidine residues (His(6)-LDH) from a crude E. coli extract. The enzyme partitioned preferentially into the top Cu-poly-VI-VCL-rich phase. After phase separation, the latter was mixed with EDTA. Temperature increase to 45 degrees C resulted in thermoprecipitation of VCL/VI-polymer, which could subsequently be recycled. His(6)-LDH remained solubilized in the aqueous phase resulting in 8-fold purification and 80% recovery in a single step.11423123