Enhancement of polyelectrolyte precipitation through the genetic fusion of charged polypeptides to enzymes
The focus of this work was to investigate the enhancement of polyelectrolyte precipitation through the genetic alteration of the target protein. The dissertation is divided into three primary chapters: a literature review of precipitation techniques which display high selectivity, experimental work including the characterization and precipitation of the fusion enzymes, and modeling of the precipitation process;The literature review of selective precipitation techniques focuses on those techniques for which relatively high purifications can be achieved. The techniques include precipitation with polyelectrolytes, affinity ligands, metal ions, and protein-binding dyes. Topics discussed include the mechanisms of selectivity and precipitation, degrees of purification which can be obtained, practical application of the precipitants, enhancement through genetic engineering, and other considerations such as mixing and recovery of the product;For the experimental work, carboxylic terminus fusions of poly(aspartic acid) were made using two proteins, [beta]-galactosidase and glucoamylase. Poly(arginine) fusions were also made with [beta]-galactosidase. The polyelectrolytes investigated were polyethyleneimine and poly(acrylic acid). Precipitation was found to be enhanced as the number of charged fusion peptides increased. An optimal tail length was observed, beyond which the addition of further peptides did not result in any further enhancement of precipitation. Activity could be quantitatively recovered from precipitates of all fusion enzymes except for the poly(arginine)-[beta]-galactosidase fusions. The degree of precipitation for each of the glucoamylase fusions was found to decrease upon increasing ionic strength. The [beta]-galactosidase control and native enzymes displayed the same behavior. The [beta]-galactosidase fusion enzymes actually displayed an increase in the degree of precipitation upon increasing the ionic strength;A model was developed to account for the presence of the charged fusion peptides in polyelectrolyte precipitation. The model is based on multiple equilibrium binding with cooperativity effects and multiple association constants. The model treats the enzyme and the fusion tail as having separate association constants. Electrostatic cooperativity is not evidenced for the binding of these negatively charged proteins to positively charged, highly branched polyethyleneimine. Experimental results for the monomeric control enzyme correlate well with model predictions. For the tailed enzymes, however, corrections for their enhanced solubility at low polyelectrolyte dosages had to be made. For the tetrameric enzyme, it is proposed that the formation of an interconnected matrix results from the multiple tails on an enzyme strongly binding to multiple polyelectrolytes. Such a mechanism would account for the increase in precipitation with increasing ionic strength if the increase in ionic strength is not sufficient to disrupt protein-polyelectrolyte binding but can reduce the electrostatic barrier to formation of a matrix of complexes carrying a net charge.