Model of the Polyelectrolyte Precipitation of Genetically Engineered Enzymes Possessing Charged Polypeptide Tails

A model is presented for the polyelectrolyte precipitation of proteins possessing charged fusion tails. The model is based on multiple equilibria binding and accounts separately for the binding of the fusion polypeptide. The predictions of the model are compared to experimental results obtained with monomeric and multimeric fusion proteins. The enzymes investigated were various fusions of glucoamylase from Aspergillus niger and β-galactosidase from Escherichia coli, respectively. Electrostatic cooperativity is not evidenced for the binding of these negatively charged proteins to positively charged, highly branched polyethyleneimine. Qualitative agreement is achieved between the model and experimental results for the behavior of the association constants of the protein and fusion polypeptide with respect to the number of polypeptide charges, ionic strength, and polymer dosage. For the precipitation of multimeric proteins, it is proposed that each of the fusion polypeptides acts as a strong electrostatic interaction site which can preferentially bind the enzyme to multiple polyelectrolytes, resulting in a tightly bound, crosslinked matrix. Increasing the ionic strength leads to a reduction in the electrostatic repulsion within the protein-polyelectrolyte complex. The combination of reduced electrostatic repulsion and the strong binding of the tails results in enhancement of the precipitation as the ionic strength is increased.