Hydrophobic interaction high-performance liquid chromatography of proteins

Goheen, S.C.; Engelhorn, Sheldon C.

doi:10.1016/s0021-9673(01)91647-4

Cited by 87 publications

(24 citation statements)

References 13 publications

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“…Proteins may change conformation in the adsorbed state under strongly retaining conditions, as indicated by multiple or misshapen chromatographic peaks (Goheen and Engelhorn, 1984;Hahn et al, 2003;Jungbauer et al, 2005;Machold et al, 2002;To et al, 2007;Wu et al, 1986a,b), and increased solvent accessibility (Buijs et al, 1999;Fogle et al, 2006;Tibbs-Jones and Fernandez, 2003;Xiao et al, 2007b). This can reduce desired product recovery (Chen et al, 2007;Kepka et al, 2005;To et al, 2007), inhibiting the application of this purification method.…”

Section: Introductionmentioning

confidence: 99%

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Deitcher

Xiao

O’Connell

et al. 2009

Biotech & Bioengineering

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A two-conformation, four-state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four-state model to the effect of salt concentration on the adsorption and unfolding of the model protein alpha-lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild-type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of alpha-lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild-type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (approximately 6-8 kJ/mol) due to increased instability of the protein. Finally, the four-state model is used to accurately predict the apparent adsorption strength of a disulfide bond-reduced variant.

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Section: Introductionmentioning

confidence: 99%

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Deitcher

Xiao

O’Connell

et al. 2009

Biotech & Bioengineering

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“…GdmSCN binds to proteins with the Gdm ϩ cation and the SCN Ϫ anion: one Gdm ϩ cation binds specifically to two peptide bonds and to aromatic side chains, 36 SCN Ϫ strongly interacts with positively charged groups in the protein. 23 For higher urea concentrations a slight decrease of retention time occurs, due to a weakening of protein-matrix interaction, according to the chromatographic behavior in HIC of proteins, previously observed. 23 In the case of Cyt C, the data in Figure 1b show that, once denatured by 4.0M GdmSCN, Cyt C chromatographic behavior is slightly affected by the composition of the mobile phase.…”

Section: Role Of Urea As Additive In the Mobile Phase Of Hic System Fmentioning

confidence: 61%

“…23 For higher urea concentrations a slight decrease of retention time occurs, due to a weakening of protein-matrix interaction, according to the chromatographic behavior in HIC of proteins, previously observed. 23 In the case of Cyt C, the data in Figure 1b show that, once denatured by 4.0M GdmSCN, Cyt C chromatographic behavior is slightly affected by the composition of the mobile phase.…”

Section: Role Of Urea As Additive In the Mobile Phase Of Hic System Fmentioning

confidence: 61%

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Characterization of denatured proteins by hydrophobic interaction chromatography: A preliminary study

et al. 2003

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In this preliminary study hydrophobic interaction chromatography (HIC) is proposed as a good tool in order to detect conformational changes induced by chemical denaturants in two globular proteins, cytochrome C (Cyt C) and myoglobin (MYO). Alterations in protein structure were manifested chromatographically by reproducible changes in peak heights, retention time, and appearance of multiple peaks. The HIC behavior of the two model proteins denatured by guanidinium thyocyanate (GdmSCN) was investigated, keeping constant various concentrations of urea in the mobile phase in a TSK-Gel Phenyl-5PW column (TosoBiosep). Suitable elution conditions provide evidence of the simultaneous presence of two denatured forms in the case of MYO, and sequential different denatured states of Cyt C.

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“…It varied from no retention in low salt to reversible or very tight binding in high salt, depending upon temperature. Organic solvents, urea, glucose, sucrose, etc., are known to reduce the strength of hydrophobic interactions and accelerate the elution of applied proteins (19,20). However, the CryIAc ␦-endotoxin, once bound at room temperature to phenyl-Sepharose matrix could not be eluted by these reagents.…”

Section: Discussionmentioning

confidence: 99%

The δ-Endotoxin Proteins Accumulate in Escherichia coli as a Protein–DNA Complex That Can Be Dissociated by Hydrophobic Interaction Chromatography

Chaturvedi

Bhakuni

Tuli

2000

Protein Expression and Purification

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Hydrophobic interaction high-performance liquid chromatography of proteins

Cited by 87 publications

References 13 publications

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Protein instability during HIC: Evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond‐reduced α‐lactalbumin variants

Characterization of denatured proteins by hydrophobic interaction chromatography: A preliminary study

The δ-Endotoxin Proteins Accumulate in Escherichia coli as a Protein–DNA Complex That Can Be Dissociated by Hydrophobic Interaction Chromatography

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