title: High-throughput protein precipitation and hydrophobic interaction chromatography: salt effects and thermodynamic interrelation.
abstract: Salt-induced protein precipitation and hydrophobic interaction chromatography (HIC) are two widely used methods for protein purification. In this study, salt effects in protein precipitation and HIC were investigated for a broad combination of proteins, salts and HIC resins. Interrelation between the critical thermodynamic salting out parameters in both techniques was equally investigated. Protein precipitation data were obtained by a high-throughput technique employing 96-well microtitre plates and robotic liquid handling technology. For the same protein-salt combinations, isocratic HIC experiments were performed using two or three different commercially available stationary phases-Phenyl Sepharose low sub, Butyl Sepharose and Resource Phenyl. In general, similar salt effects and deviations from the lyotropic series were observed in both separation methods, for example, the reverse Hofmeister effect reported for lysozyme below its isoelectric point and at low salt concentrations. The salting out constant could be expressed in terms of the preferential interaction parameter in protein precipitation, showing that the former is, in effect, the net result of preferential interaction of a protein with water molecules and salt ions in its vicinity. However, no general quantitative interrelation was found between salting out parameters or the number of released water molecules in protein precipitation and HIC. In other words, protein solubility and HIC retention factor could not be quantitatively interrelated, although for some proteins, regular trends were observed across the different resins and salt types.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands.
authors.forename: Beckley K
authors.initials: BK
authors.lastname: Nfor
authors.forename: Nienke N
authors.initials: NN
authors.lastname: Hylkema
authors.forename: Koenraad R
authors.initials: KR
authors.lastname: Wiedhaup
authors.forename: Peter D E M
authors.initials: PD
authors.lastname: Verhaert
authors.forename: Luuk A M
authors.initials: LA
authors.lastname: van der Wielen
authors.forename: Marcel
authors.initials: M
authors.lastname: Ottens
mesh_terms: D000818:Animals; D011232:Chemical Precipitation; D002853:Chromatography, Liquid / Q000295:instrumentation* / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D011506:Proteins / Q000737:chemistry*; D012492:Salts / Q000737:chemistry*; D012685:Sepharose / Q000737:chemistry
chemical_list: D011506:Proteins; D012492:Salts; D012685:Sepharose