Metal affinity protein precipitation: Effects of mixing, protein concentration, and modifiers on protein fractionation

✍ Scribed by Harish V. Iyer; Todd M. Przybycien

• Publisher: John Wiley and Sons
• Year: 1995
• Tongue: English
• Weight: 883 KB
• Volume: 48
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/bit.260480405

✦ Synopsis

Previous work by u s and others h a s shown that mixing impacts apparent protein solubility in single protein precipitations. In this work, w e probe the effects of contacting conditions on fractional precipitation behavior at the bench scale. We have chosen metal affinity precipitation a s o u r model system; the kinetics of this mode of precipitation are very rapid and largely irreversible and, consequently, mixing conditions govern the extent of fractionation and purity of the product in such a process. Our experimental strategy involved a three-pronged approach to control t h e effects of contacting conditions on precipitate yield, purity, and particle size distribution. First, w e studied the impact of process variables that control precipitant concentrations in the reactor including impeller speed and precipitant addition rate. Second, we controlled the rate of precipitation by changing the initial protein concentration to alter the protein-protein collision rate. Third, w e examined the role of the rnolecular-level kinetics of affinity precipitation by using modifiers that compete with surface moieties to bind the metal ion, thereby reducing its availability. Our model process and protein system consisted of zinc precipitations of mixtures of bovine serum albumin and bovine y-globulins, carried out at a nominal I -L scale; glycine w a s examined a s a modifier. Faster impeller speeds and lower precipitant addition rates increased the desired protein yields, decreased purities, and reduced average precipitate particle size. Higher initial protein concentrations were found to produce precipitates with higher yields, lower purities and diminished particle sizes. Experiments with glycine indicated that modifiers in the precipitant solution serve to increase product purity, decrease yield, and increase the average particle size in bench-scale precipitations.