Utilization of Lysozyme Charge Ladders to Examine the Effects of Protein Surface Charge Distribution on Binding Affinity in Ion Exchange Systems

Chung, Wai Keen; Evans, Steven T.; Freed, Alexander S.; Keba, James J.; Baer, Zachary C.; Rege, Kaushal; Cramer, Steven M.

doi:10.1021/la902135t

Cited by 28 publications

(22 citation statements)

References 26 publications

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“…The retention mechanism of lysozyme in cation‐exchange chromatography has been thoroughly investigated. By using an artificial lysozyme protein charge ladder, Chung et al identified two binding regions, which consist of five arginines, two lysines, one histidine, and the N‐terminus . Our results for the number of amino acids involved in lysozyme binding to cation exchanger are in a good agreement with these findings.…”

Section: Resultssupporting

confidence: 88%

Modeling of salt and pH gradient elution in ion-exchange chromatography

2014

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The separation of proteins by internally and externally generated pH gradients in chromatofocusing on ion-exchange columns is a well-established analytical method with a large number of applications. In this work, a stoichiometric displacement model was used to describe the retention behavior of lysozyme on SP Sepharose FF and a monoclonal antibody on Fractogel SO3 (S) in linear salt and pH gradient elution. The pH dependence of the binding charge B in the linear gradient elution model is introduced using a protein net charge model, while the pH dependence of the equilibrium constant is based on a thermodynamic approach. The model parameter and pH dependences are calculated from linear salt gradient elutions at different pH values as well as from linear pH gradient elutions at different fixed salt concentrations. The application of the model for the well-characterized protein lysozyme resulted in almost identical model parameters based on either linear salt or pH gradient elution data. For the antibody, only the approach based on linear pH gradients is feasible because of the limited pH range useful for salt gradient elution. The application of the model for the separation of an acid variant of the antibody from the major monomeric form is discussed.

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Section: Resultssupporting

confidence: 88%

Modeling of salt and pH gradient elution in ion-exchange chromatography

2014

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“…Proteins can interact with a chromatographic surface via a preferred binding face or through multiple binding sites and orientations (Chung et al, ). In order to evaluate the relative importance of the four proposed multimodal binding sites (I–IV) identified on the wild type as shown in Figure , we first designed and produced a set of Fab charge variants (Table , C1 − to C5 − ) with reduced positive EP in each site.…”

Section: Resultsmentioning

confidence: 99%

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Karkov

Krogh

Woo

et al. 2015

Biotech & Bioengineering

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In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.

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“…Protein charge ladders were generated by acylation of the lysine ɛ‐amino groups of either lysozyme ( r p = 2 nm, 14.3 kDa, pI = 11) or α‐lactalbumin ( r p = 2 nm, 14.2 kDa, pI = 4.6) using acetic anhydride (Sigma 242845) following the procedure described by Chung et al (Figure ). The acylation reaction converts the protonatable amino group into a neutral amide, thereby reducing the number of positive charge groups on the protein.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the effects of electrostatic interactions on protein transport through zwitterionic ultrafiltration membranes using protein charge ladders

Hadidi

Zydney

2014

J of Applied Polymer Sci

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A large number of studies have shown that zwitterionic ultrafiltration membranes have very low protein fouling due to the strongly hydrated structure of the zwitterionic modification. However, there is much less known about the effects of the zwitterionic functionality on the electrostatic interactions governing protein transport through these membranes. The objective of this work was to use protein charge ladders to evaluate the effects of electrostatic interactions on protein transport through cellulosic ultrafiltration membranes modified with a zwitterionic functionality. Data were obtained using protein charge ladders formed by reacting lysozyme and α‐lactalbumin with acetic anhydride to generate a series of protein derivatives (ladders) differing by single charge groups but with essentially identical size. Protein retention was greater for the more positively charged elements within the charge ladder, consistent with a weak electrostatic repulsion from the zwitterionic membrane which had a small positive charge at pH 7. Data for all elements of the protein charge ladder (both positively and negatively charged) were in excellent agreement with calculations of a theoretical model based on the partitioning of a charged sphere (protein) in a charged cylinder. The results demonstrate the potential of using zwitterionic membranes for enhanced ultrafiltration with high selectivity and minimal fouling. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41540.

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Utilization of Lysozyme Charge Ladders to Examine the Effects of Protein Surface Charge Distribution on Binding Affinity in Ion Exchange Systems

Cited by 28 publications

References 26 publications

Modeling of salt and pH gradient elution in ion-exchange chromatography

Modeling of salt and pH gradient elution in ion-exchange chromatography

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants

Analysis of the effects of electrostatic interactions on protein transport through zwitterionic ultrafiltration membranes using protein charge ladders

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