N-Terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition

Fexby, Sara; Ihre, Henrik; Alstine, James Van; Bülow, Leif

doi:10.1016/j.jchromb.2004.01.049

Cited by 9 publications

(5 citation statements)

References 47 publications

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“…Media 6 was widely used commercial Phenyl Sepharose HP. The commercial and experimental media show good HIC performance (Fexby et al, 2004) (Maloisel and Thevenin, 2004;Van Alstine et al, 2004).…”

Section: Chromatographic Mediamentioning

confidence: 99%

“…The volume of the mobile phase was taken to be the sum of the supernatant volume (added and in original slurry) together with interparticle voids. This volume was used to compute the supernatant protein Sepharose media with moderately hydrophobic polymeric coating formed in situ using radical initiated grafting of hydroxybutylvinyl ether monomer Ihre (2003); Fexby et al (2004) 4,5 Sepharose media with more hydrophobic experimental ligands Maloisel and Thevenin (2004) 6 Phenyl Sepharose High Performance HIC media Commercially available from GE Healthcare concentration (C sup ). The number of moles in the adsorbed phase was determined by mass balance.…”

Section: Batch Adsorption Measurementsmentioning

confidence: 99%

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Protein instability during HIC: Hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects

Xiao

Jones

Laurent

et al. 2006

Biotech & Bioengineering

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Unfolding of marginally stable proteins is a significant factor in commercial application of hydrophobic interaction chromatography (HIC). In this work, hydrogendeuterium isotope exchange labeling has been used to monitor protein unfolding on HIC media for different stationary phase hydrophobicities and as a function of ammonium sulfate concentration. Circular dichroism and Raman spectroscopy were also used to characterize the structural perturbations experienced by solution phase protein that had been exposed to media and by protein adsorbed on media. As expected, greater instability is seen on chromatographic media with greater apparent hydrophobicity. However, increased salt concentrations also led to more unfolding, despite the well-known stabilizing effect of ammonium sulfate in solution. A thermodynamic framework is proposed to account for the effects of salt on both adsorption and stability during hydrophobic chromatography. Using appropriate estimates of input quantities, analysis with the framework can explain how salt effects on stability in chromatographic systems may contrast with solution stability. Biotechnol. Bioeng. 2007;96: 80-93.

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Section: Chromatographic Mediamentioning

confidence: 99%

Section: Batch Adsorption Measurementsmentioning

confidence: 99%

Protein instability during HIC: Hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects

Xiao

Jones

Laurent

et al. 2006

Biotech & Bioengineering

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“…The following Eqs. (9)(10)(11) determine the position of the last chromatographic step in the sequence of K M steps:…”

Section: Constraint Equations For Tag Selectionmentioning

confidence: 99%

“…It has been reported that the addition of specific tags (7)(8)(9)(10) improves the performance of purification stages. Examples of these tags are: poly-His-tag, poly-Arg-tag, calmodulin-binding peptide (CBP), cellulase-binding domain (CBD), DsbA, c-myc-tag, glutathione S-transferase (GST), FLAG-tag, HAT-tag, maltose-binding protein (MBP), NusA, S-tag, SBP-tag, Strep II-tag, thioredoxin, Biotin acceptor peptide (BAP), and many others.…”

Section: Introductionmentioning

confidence: 99%

“…Examples of these tags are: poly-His-tag, poly-Arg-tag, calmodulin-binding peptide (CBP), cellulase-binding domain (CBD), DsbA, c-myc-tag, glutathione S-transferase (GST), FLAG-tag, HAT-tag, maltose-binding protein (MBP), NusA, S-tag, SBP-tag, Strep II-tag, thioredoxin, Biotin acceptor peptide (BAP), and many others. (6,11); short hydrophobic peptide tags, e.g., (WP)2, (WP)4, (Y)3(YP)3, (Y)3(P)2, (Y)4, (YP)4, (Y)6, (Y)6(P)2, (Y) 8 have been also used (7,9,10). The use of polypeptide tags has many advantages, such as a. fewer genetic modifications in the target product; b. they have a minimum impact on the tertiary structure and biological activity of the fusion protein because of their small size; c. it is relatively easy to remove the tag (a specific cleavage location may be included).…”

Section: Introductionmentioning

confidence: 99%

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Experimental Validation of the Predictions of a Mathematical Model for Protein Purification and Tag Selection

Lienqueo

Shene

Quiroga

et al. 2010

Separation Science and Technology

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To improve downstream processing, protein engineering can be used to modify particular properties of a protein, such as specific affinity, charge, or hydrophobicity. The most common modification involves the peptide tags fusion to the protein. Nevertheless, the selection of both the optimal peptide tag and the right purification system to use is not trivial. The aim of this work is to experimentally validate our previous mathematical model based on MINLP models. This model was modified to find the minimum number of chromatographic operations, and the ideal tag for obtaining a required level of purification. A weighted linear combination of the number of purification steps and the target protein purity obtained after the last step was used as the objective function. The chromatographic steps suggested by the model were carried out using an example based on two mutated cutinases. The results show that average deviations between experimental data and those predicted by the model, for yield and purity, are ca. 15%, confirming that model predictions are reliable and could be used for selecting the best purification processes without experimental tests.

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Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

Kepka

Collet

Roos

et al. 2005

Journal of Chromatography A

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N-Terminal tagged lactate dehydrogenase proteins: evaluation of relative hydrophobicity by hydrophobic interaction chromatography and aqueous two-phase system partition

Cited by 9 publications

References 47 publications

Protein instability during HIC: Hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects

Protein instability during HIC: Hydrogen exchange labeling analysis and a framework for describing mobile and stationary phase effects

Experimental Validation of the Predictions of a Mathematical Model for Protein Purification and Tag Selection

Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

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