AQUEOUS 2-PHASE SYSTEM FORMED BY THERMOREACTIVE VINYL IMIDAZOLE VINYLCAPROLACTAM 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 a pplication combining two attractive concepts in downstream processing: the immobilised metal affinity partitioning and the use of thermoprec ipitating polymers. ATPS consisting of the thermoprecipitating copolym er of N-vinyl caprolactam/1-vinyl imidazole loaded with Cu ions (Cu-po ly-VI-VCL) in the top phase and dextran T70 in the bottom phase was us ed for purification of recombinant lactate dehydrogenase carrying an a ffinity 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 EDT A. Temperature increase to 45 degrees C resulted in thermoprecipitatio n of VCL/VI-polymer, which could subsequently be recycled. His(6)-LDH remained solubilized in the aqueous phase resulting in 8-fold purifica tion and 80% recovery in a single step.