Non-native aggregation of anti-streptavidin immunoglobulin Gamma-1
Date
2012
Authors
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Publisher
University of Delaware
Abstract
Global aggregation behavior of a model IgG1 protein, anti-streptavidin (antiSA), was characterized as a function of pH and [NaCl] in acidic conditions. The relative rate of aggregation as illustrated by T2h (temperature at which isothermal incubation resulted in an aggregation half-life of two hours), and mechanisms of aggregation at the different solution conditions were monitored. It was found that relative rate of aggregation is strongly influenced by conformational stability of the protein but generally independent of colloidal interactions. A state diagram of aggregation mechanisms suggested that the mechanism of aggregate growth, and thus the resulting aggregate type, is strongly dependent on electrostatic, inter-molecular interactions. When this state diagram was overlayed with a phase diagram from reversible phase transition studies, it was found that there was a strong correlation between the two, suggesting that the formation of insoluble aggregates from kinetic and thermodynamic means are by the same or similar mechanisms. Several structural assays (far-UV CD, and fluorescence (intrinsic and ThT binding)) were used to analyze the changes in secondary and tertiary structure due to aggregation by the various mechanisms. It was found that ThT bound equally to all types of soluble aggregates, suggesting amyloid fibril formation at all conditions observed. However, the other assays showed varying amounts of structural changes depending on pH and NaCl concentrations, with conditions that have largest inter-and intra-molecular repulsions having the largest change in structure.This suggested that a combination of conformation change and electrostatic interactions may influence the type of aggregates that form via thermal stress.