The development of chromatography for the characterization of protein interactions: a personal perspective

Winzor, Donald J.

doi:10.1042/bst0311010

Cited by 7 publications

(3 citation statements)

References 32 publications

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“…Indeed, the c(s) distributions can exhibit broadening for other reasons. Nevertheless, just as our analysis of the difference curves by incorporating kinetics, so too can the gel filtration elution profiles for ClpB be analyzed by incorporating kinetic parameters . However, to our knowledge, this analysis has not been applied to the examination of ClpB using gel filtration.…”

Section: Discussionmentioning

confidence: 99%

Examination of the dynamic assembly equilibrium for E. coli ClpB

Lin

Lucius

2015

Proteins

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Escherichia coli ClpB is a heat shock protein that belongs to the AAA+ protein superfamily. Studies have shown that ClpB and its homologue in yeast, Hsp104, can disrupt protein aggregates in vivo. It is thought that ClpB requires binding of nucleoside triphosphate to assemble into hexameric rings with protein binding activity. In addition, it is widely assumed that ClpB is uniformly hexameric in the presence of nucleotides. Here we report, in the absence of nucleotide, that increasing ClpB concentration leads to ClpB hexamer formation, decreasing NaCl concentration stabilizes ClpB hexamers, and the ClpB assembly reaction is best described by a monomer, dimer, tetramer, hexamer equilibrium under the three salt concentrations examined. Further, we found that ClpB oligomers exhibit relatively fast dissociation on the time scale of sedimentation. We anticipate our studies on ClpB assembly to be a starting point to understand how ClpB assembly is linked to the binding and disaggregation of denatured proteins.

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Section: Discussionmentioning

confidence: 99%

Examination of the dynamic assembly equilibrium for E. coli ClpB

Lin

Lucius

2015

Proteins

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“…Frontal analysis using soluble proteins Another common technique for examining drug-protein interactions in solution is frontal analysis. 7,[45][46][47][48][49][50][51] This approach was first employed in 1964-1965 with soluble agents to examine protein-protein binding 45 and drug-protein interactions 46 and is still an important method for such measurements. Frontal analysis using soluble proteins is different from the zonal elution methods described in the previous section because a large volume of a drug/protein mixture is used instead of a small plug of sample.…”

Section: Zonal Elution Methods Using Soluble Proteinsmentioning

confidence: 99%

Chromatographic analysis of drug interactions in the serum proteome

et al. 2011

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The binding of drugs with serum proteins and binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drug-protein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems.

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“…Various chromatography based techniques have emerged in the last years in order to characterize protein-protein interactions [17,[26][27][28][29][30][31]. For examples, Stevens [32] proposed the use of small-zone size exclusion chromatography for investigating protein-protein association and Wen et al [33] showed the benefit of the additional combination of various detection methods for analyzing protein-protein interaction (see also [17]).…”

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confidence: 99%

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

Brun¹,

Frieß²,

Schultz-Fademrecht

et al. 2009

Biotechnology Journal

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The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein‐protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B22), a parameter of solution non‐ideality, which is obtained using self‐interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B22 was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B22 is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B22 decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein‐protein interactions. The B22 parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting‐in/salting‐out effect. It is also shown that B22 correlates with protein solubility as well as physical protein stability.

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The development of chromatography for the characterization of protein interactions: a personal perspective

Cited by 7 publications

References 32 publications

Examination of the dynamic assembly equilibrium for E. coli ClpB

Examination of the dynamic assembly equilibrium for E. coli ClpB

Chromatographic analysis of drug interactions in the serum proteome

Lysozyme‐lysozyme self‐interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

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