Investigation of protein binding affinity and preferred orientations in ion exchange systems using a homologous protein library

Abstract

A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure-property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems.

Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis

“…One explanation for this result is that, relative to n-PTEN, 4p-PTEN undergoes a conformational change which buries its negatively charged C-tail, reducing its availability for interacting with cationic resin. The fact that clusters of charges can be more important than overall charge for protein–ion exchange resin interactions has been discussed previously (Chung et al, 1989; Hou et al, 2010).

10.7554/eLife.00691.013Figure 4.Conformational changes associated with PTEN phosphorylation.( A ) With a gradient of 0–50% NaCl over 250 ml on an anion exchange column, 4p-PTEN elutes at ∼70 ml while n-PTEN elutes at ∼100 ml.

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Resolution of heterogeneous charged antibody aggregates via multimodal chromatography: A comparison to conventional approaches

“…One potential explanation for the difficulty in removal of dimers using cation exchange chromatography is that the distribution of charge on the dimers may be composed of larger acidic or negative charge patches compared to the monomer. The importance of the charge distribution as well as the pI of a protein impacting chromatographic retention has been previously described in the literature . Cation and anion exchange chromatography results showed higher order aggregates have a higher net charge and a different, basic charge distribution than the dimers and monomer and are easily removed using an ion exchange modality.…”

Purification design and practice for pertactin, the third component of acellular pertussis vaccine, from Bordetella pertussis

“…Separation by IEC is achieved on the basis of differences in charge properties of those ionizable molecules. Thus effective design of the process can be possible with better understanding of surface charge properties of target protein, regarding that charge clusters in addition to local protein geometry have been proved critical for protein retention on IEC [23,24]. We conducted analysis of protein surface potential property for selection of appropriate purification methods.…”