Abstract
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-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°C resulted in thermoprecipitation of VCL/VI-polymer, which could subsequently be recycled. His-LDH remained solubilized in the aqueous phase resulting in 8-fold purification and 80 % recovery in a single step.
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Teixeira Franco, T., Galaev, I., Hatti-Kaul, R. et al. Aqueous two-phase system formed by thermoreactive vinyl imidazole/vinyl caprolactam copolymer and dextran for partitioning of a protein with a polyhistidine tail. Biotechnology Techniques 11, 231–236 (1997). https://doi.org/10.1023/A:1018430319981
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DOI: https://doi.org/10.1023/A:1018430319981