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 equil ibria binding and accounts separately for the binding of the fusion po lypeptide. The predictions of the model are compared to experimental r esults obtained with monomeric and multimeric fusion proteins. The enz ymes investigated were various fusions of glucoamylase from Aspergillu s niger and beta-galactosidase from Escherichia coli, respectively. El ectrostatic cooperativity is not evidenced for the binding of these ne gatively charged proteins to positively charged, highly branched polye thyleneimine. 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 polyp eptide charges, ionic strength, and polymer dosage. For the precipitat ion of multimeric proteins, it is proposed that each of the fusion pol ypeptides acts as a strong electrostatic interaction site which can pr eferentially bind the enzyme to multiple polyelectrolytes, resulting i n a tightly bound, crosslinked matrix. Increasing the ionic strength l eads to a reduction in the electrostatic repulsion within the protein- polyelectrolyte complex. The combination of reduced electrostatic repu lsion and the strong binding of the tails results in enhancement of th e precipitation as the ionic strength is increased.