One step purification of chymosin by mixed mode chromatography

Burton, S. C.; Haggarty, N. W.; Harding, David

doi:10.1002/(sici)1097-0290(19971005)56:1<45::aid-bit5>3.3.co;2-u

Cited by 27 publications

(42 citation statements)

References 26 publications

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“…16 The aspartic protease chymosin has its highest activity at pH 3-4 and it is the only enzyme known to specifically cleave the Phe 105 -Met 106 peptide bond in j-casein, resulting in separation of j-casein. 17,18 This process is commonly used in cheesemaking.…”

Section: Introductionmentioning

confidence: 99%

The enzyme-mediated autodeposition of casein: effect of enzyme immobilization on deposition of protein structures

2016

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The enzyme-mediated autodeposition on the example of casein is reviewed, and deposition of casein structures is investigated in relation to applied immobilization methods of enzyme. First, casein is described in detail with respect to its structure in aqueous environments, followed by presentation of historical and current nonfood applications of casein. The presented process uses enzymes to trigger deposition of bio-based particles in close proximity to a support surface and allows for a high control over film formation and site-specificity. This is ensured by immobilization of enzyme on the support material. The herein described system is based on casein as protein and chymosin as enzyme. Different immobilization methods are investigated with respect to obtainable casein coatings, layers, and structures. Physical adsorption of enzyme enables the formation of casein coatings with controllable film thickness and is suitable for in situ buildup of adhesive layers due to diffusion of enzyme. The highest control over film formation is provided by covalent attachment of enzyme. Based on the attained results, the enzymemediated autodeposition gives new insights into biological material design. Graphical abstractEnzyme Casein micelle intact Bulk zone (no enzyme) Reaction zone Phe Met Support Support Casein micelle cleaved

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Section: Introductionmentioning

confidence: 99%

The enzyme-mediated autodeposition of casein: effect of enzyme immobilization on deposition of protein structures

2016

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“…Recent advances in the design of multimodal (MM) chromatographic systems have produced previously undescribed classes of chromatographic materials that can provide alternative and improved selectivities as compared to traditional single mode chromatographic materials (3)(4)(5)(6)(7)(8)(9). Johansson et al have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions (4,5).…”

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confidence: 99%

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

Chung

Freed

Holstein

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarsegrained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems.ligand binding site | mixed mode chromatography | protein-ligand interactions | binding site mapping | pseudoaffinity T he development of efficient bioseparation processes for the production of high-purity biopharmaceuticals is one of the most pressing challenges facing the pharmaceutical and biotechnology industries today. In addition, high-resolution separations for complex bioanalytical applications are becoming increasingly important. Although it is generally accepted that nonspecific interactions can often complicate single mode chromatographic separations (e.g., ion-exchange, reversed-phase), these additional interactions can also result in unexpected selectivities (1, 2).Recent advances in the design of multimodal (MM) chromatographic systems have produced previously undescribed classes of chromatographic materials that can provide alternative and improved selectivities as compared to traditional single mode chromatographic materials (3-9). Johansson et al. have developed a library of MM ligands that can be employed for the capture of charged proteins under high salt conditions (4, 5). Liu et al. have developed a silica-based MM resin capable of weak anion-exchange and reversed-phase interactions for the simultaneous separation of acidic, basic, and neutral pharmaceutical compounds (9). Small ligand pseudoaffinity chromatographic materials such as those used for hydrophobic charge induction chromatography have resulted in previously undescribed classes of MM ligands that offer unique selectivities due more to multiple low affinity MM interactions than to specific binding to certain classes of proteins (10). In addition, several libraries of MM ligands have been recently developed and employed on chro...

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“…HCIC matrices have a higher ligand density than HIC, therefore, it could bind proteins at low ionic strength. High ligand density (80 mmol/mL) matrices have been used for mixed mode hydrophobic ionic chromatography for the purification of chymosin, which resulted in high capacity (Burton, 1997). Furthermore, chymosin could be adsorbed at high and low ionic strength, therefore, a pretreatment step of salt addition, or removal by dialysis, dilution or ultrafiltration was not required.…”

Section: The Mechanism Of Hcicmentioning

confidence: 99%