Rapid Measurement of Protein Osmotic Second Virial Coefficients by Self-Interaction Chromatography

Abstract

Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein.

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

“…This is the case when assuming no changes in protein conformation as well as neglecting higher-order processes [53]. Using relative low protein surface coverages, Teske et al [80] The data presented in this study are in accordance to the SIC results presented by Tessier et al [66]. They have also shown a quantitative agreement between virial coefficients measured by SIC and by static light scattering.…”

“…At 0.8 M salt concentration one observed: (B 22 > 0) (see Figure 6B2), which is representative for repulsive interactions. This trend has also been described by Tessier et al [66]. The reason for this behavior is due to binding of the divalent magnesium cation to the acidic residues of lysozyme.…”

“…This is especially observed after the column has been stored for 161 days. Based on the obtained data, stability of the lysozyme functionalized column, under the chosen storage conditions, is given for 3 months, which is in accordance to Tessier et al [66]. This allows SIC as being used as a high throughput screening method for the evaluation of a large number of solution conditions.…”

Molecular thermodynamics for some applications in biotechnology

“…In a separate experiment, the same procedure is used except that now there is no immobilized protein 2 on the column; that experiment gives retention time t ro . The key experimental quantity is the reduced relative retention time (retention factor) k : Figure 12 shows some results for obtained by Teske (26) for an aqueous two-protein system k ι containing ovalbumin (2) and lysozyme (2). Results at 25 o C are shown as a function of pH for two cases: first, for an aqueous solvent containing 0.1m ionic strength sodium chloride and second, for an aqueous solvent containing 0.1m ionic strength magnesium chloride.…”

Effect of the ion-protein dispersion interactions on the protein-surface and protein-protein interactions

“…This is because it can be measured using traditional colloidal characterization techniques like static and dynamic light scattering, 7,9,10 membrane osmometry, 11,12 sedimentation, 13 and chromatographic methods. [14][15][16] However, these techniques have some limitations. They are generally labor-intensive and expensive in terms of time and protein cost.…”