RECCR Rensselaer Exploratory Center for Cheminformatics Research


Molecular Lipophilicity Potential



PI:Curt M. Breneman

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Protein Dissimilarity Analysis using Shape/Property Descriptors

Principal Investigator: Curt M. Breneman

Professor, Department of Chemistry and Chemical Biology Rensselaer Polytechnic Institute

Hydrophobic interaction chromatography (HIC) is an important bioseparation technique for protein purification, it is based on the reversible interaction between the hydrophobic patches on protein molecules and the hydrophobic surface of the stationary phase. The stationary phase consists of small non-polar groups (butyl, octyl or phenyl) attached to a hydrophilic polymer backbone such as cross-linked dextran or agarose. Separations by HIC are often designed using nearly opposite conditions to those used in ion exchange chromatography. The sample is loaded in a buffer containing a high concentration of a non-denaturing salt like ammonium sulfate. The proteins are then eluted as the concentration of the salt in the buffer is decreased. HIC is widely used in the downstream processing of proteins as it provides an alternative basis for selectivity compared with ion-exchange and other modes of adsorption. Additionally, HIC is an ideal “next step” after precipitation with ammonium sulfate or elution in high salt during ion-exchange chromatography (IEC) (Shukla et al., 2000).

Several factors influence the efficiency of separation process in HIC systems, such as protein hydrophobicity, protein size (Fausnaugh et al., 1984), type of stationary phase resin (Erikkson et al., 1998), type and concentration of salt (Sofer et al., 1998), buffer pH, temperature and mode of operation (e.g. gradient, displacement, etc.). Despite efforts towards understanding the retention mechanism of proteins in HIC systems, none of the proposed theories has general acceptance (Melander et al., 1977; Melander et al., 1984; Staby et al., 1996; Jennissen, 1986), the selection of appropriate chromatographic conditions for the separation of complex biological mixtures in HIC remains a challenge.


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