Recovery of a charged-fusion protein from cell extracts by polyelectrolyte precipitation

Beta-galactosidase served as a model system to explore the feasibility of enhancing the selectivity of a low-cost, easily scaled separation method-precipitation. Enhanced selectivity was sought by fusing the enzyme with polypeptide tails including 5 and 11 aspartates. The unfused protein could not be selectively removed from the Escherichia coli cell extract by precipitation with polyethylenimine (PEI), but the longest fusion could be selectively removed. The presence of nucleic acids limited the purification attainable. Pretreatment with nuclease followed by diafiltration resulted in an extract from which the same fusion could be precipitated with greater than fivefold enrichment, while the untailed enzyme remained unenriched by the same precipitation step. Selectivity is attributed to the binding strength of the polyanionic tails to the polycationic PEI.