Purification of Glutamate Dehydrogenase from Liver and Brain

Motherway, Martha; Tipton, Keith F.; McCarthy, Alun D.; Couée, Ivan; Irwin, Jane A.

doi:10.1002/0471140864.ps0104s29

Cited by 4 publications

(3 citation statements)

References 42 publications

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“…Our technique builds upon other technologies in which proteins can be immobilized and retain their ability to participate in molecular recognition events. For example, the exploitation of immobilized proteins pervades modern separation techniques (e.g., affinity chromatography, immunoprecipitation, Western blotting), sensor and cell binding biophysical studies (enzyme-linked immunosorbant assay (ELISA), – surface plasmon resonance (SPR), blot rolling, , and cell-free bead adhesion assays). In all of these examples, proteins in solution are affixed to the substrate by sandwich immobilization techniques or by physisorption or chemisorption.…”

Section: Resultsmentioning

confidence: 99%

Multifunctional Surfaces with Discrete Functionalized Regions for Biological Applications

et al. 2008

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In this paper we describe a method for creating multifunctional glass surfaces presenting discrete patches of different proteins on an inert PEG-functionalized background. Microcontact printing is used to stamp the substrate with octadecyltrichlorosilane to define the active regions. The substrate is then back-filled with PEG-silane {[[2-methoxypoly(ethyleneoxy)]propyl]trimethoxysilane} to define passive regions. A microfluidics device is subsequently affixed to the substrate to deliver proteins to the active regions, with as many channels as there are proteins to be patterned. Examples of trifunctional surfaces are given which present three terminating functional groups, i.e., protein 1, protein 2, and PEG. These surfaces should be broadly useful in biological studies, as patch size is well established to influence cell viability, growth, and differentiation. Three examples of cellular interactions with the surfaces are demonstrated, including the capture of cells from a single cell suspension, the selective sorting of cells from a mixed suspension, and the adhesion of cells to ligand micropatches at critical shear stresses. Within these examples, we demonstrate that the patterned immobilized proteins are active, as they retain their ability to interact with either antibodies in solution or receptors presented by cells. When appropriate (e.g., for E-selectin), proteins are patterned in their physiological orientations using a sandwich immobilization technique, which is readily accommodated within our method. The protein surface densities are highly reproducible in the patches, as supported by fluorescence intensity measurements. Potential applications include biosensors based on the interaction of cells or of marker proteins with protein patches, fundamental studies of cell adhesion as a function of patch size and shear stress, and studies of cell differentiation as a function of surface cues.

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Section: Resultsmentioning

confidence: 99%

Multifunctional Surfaces with Discrete Functionalized Regions for Biological Applications

et al. 2008

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“…However, the system used may have more fundamental effects on the enzyme behaviour by affecting their conformational motions [ 24 ]. Furthermore, the buffer components used may have differential effects; for example, phosphate and potassium ions activate some enzymes, but inhibit others [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 ], and purified ‘native’ glutamate dehydrogenase is stable in phosphate buffer, but not in Tris buffer [ 28 ], whereas Tris and HEPES inhibit carbamoyl synthase (ammonia) [ 29 ].…”

Section: Selecting Appropriate Assay Conditionsmentioning

confidence: 99%

Parameter Reliability and Understanding Enzyme Function

McDonald

Tipton

2022

Molecules

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Knowledge of the Michaelis–Menten parameters and their meaning in different circumstances is an essential prerequisite to understanding enzyme function and behaviour. The published literature contains an abundance of values reported for many enzymes. The problem concerns assessing the appropriateness and validity of such material for the purpose to which it is to be applied. This review considers the evaluation of such data with particular emphasis on the assessment of its fitness for purpose.

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“…As discussed in previous sections, much can be done to optimize the stability of Ig domains by manipulation of the polypeptide sequences of Ab fragments. Yet, the capacity of osmolytes to promote protein folding, packing, and conformation stability provides additional strategies to prevent the aggregation of Ab based proteins during the different stages of their production, distribution, storage, and usage [ 83 – 85 ]. Osmolytes like betaine, glycerol, glycine and proline have been used to optimize the yield of different recombinant proteins and immunotoxins synthetized in E. coli as soluble native products [ 76 , 86 ].…”

Section: Use Of Osmolytes To Prevent Ig Unfolding and Aggregationmentioning

confidence: 99%

Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments

Gil

Schrum

2013

ABB

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Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacokinetic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding; 2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig domains when pursuing the development of Ab fragment-based biotechnologies.

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Purification of Glutamate Dehydrogenase from Liver and Brain

Cited by 4 publications

References 42 publications

Multifunctional Surfaces with Discrete Functionalized Regions for Biological Applications

Multifunctional Surfaces with Discrete Functionalized Regions for Biological Applications

Parameter Reliability and Understanding Enzyme Function

Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments

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