Proteins hydrophobic interaction chromatography

Reversed-phase chromatography is widely used as an analytical tool for protein chromatography, but it is not as commonly found on a process scale for protein purification because the solvents which make up the mobile phase, ie, acetonitrile, isopropanol, methanol, and ethanol, reversibly or irreversibly denature proteins. Hydrophobic interaction chromatography appears to be the least common process chromatography tool, possibly owing to the relatively high costs of the salts used to make up the mobile phases. 

Fausnaugh, J.L. Regnier, F.E. Solute and mobile phase contributions to retention in hydrophobic interaction chromatography of proteins. J. Chromatogr. 1986, 359, 131. Wetlaufer, D.B. Koenigbauer, M.R. Surfactant-mediated protein hydrophobic-interaction chromatography. J. Chromatogr. 1986, 359, 55. 

The differences in sizes and locations of hydrophobic pockets or patches on proteins can be exploited in hydrophobic interaction chromatography (HIC) and reversed-pha.se chromatography (RPC) discrimination is based on interactions between the exposed hydro-... 

The protein can be further purified by hydrophobic interaction chromatography on a column of Butyl Sepharose 4 Fast Flow (Pharmacia elution with decreasing concentration of (NH4)2S04 starting at 1.5 M), and gel filtration on a column of Superdex 200 Prep (Pharmacia Inouye et al., 2000). 

Owing to the weak hydrophobicity of the PEO stationary phases and reversibility of the protein adsorption, some advantages of these columns could be expected for the isolation of labile and high-molecular weight biopolymers. Miller et al. [61] found that labile mitochondrial matrix enzymes — ornitine trans-carbomoylase and carbomoyl phosphate synthetase (M = 165 kDa) could be efficiently isolated by means of hydrophobic interaction chromatography from the crude extract. 

Separations in hydrophobic interaction chromatography have been modeled as a function of the ionic strength of the buffer and of the hydrophobicity of the column, and tested using the elution of lysozyme and ovalbumin from octyl-, butyl- and phenyl-Sepharose phases.2 The theoretical framework used preferential interaction analysis, a theory competitive to solvophobic theory. Solvophobic theory views protein-surface interaction as a two-step process. In this model, the protein appears in a cavity in the water formed above the adsorption site and then adsorbs to the phase, with the free energy change... 

Perkins, T.W., Mak, D.S., Root, T.W., and Lightfoot, E.N., Protein retention in hydrophobic interaction chromatography modeling variation with buffer ionic strength and column hydrophobicity, J. Chromatogr. A, 766, 1, 1997. 

Xie, S., Svec, F., and Frechet, J.M.J., Rigid porous polyacrylamide-based monolithic columns containing butyl methacrylate as a separation medium for the rapid hydrophobic interaction chromatography of proteins,. Chromatogr. A, 775, 65, 1997. 

Wu, S.-L., Benedek, K., and Karger, B. L., Thermal behavior of proteins in high-performance hydrophobic-interaction chromatography, /. Chromatogr., 359, 3, 1986. 

Protein separation by hydrophobic interaction chromatography is dependent upon interactions between the protein itself, the gel matrix and the surrounding aqueous solvent. Increasing the ionic strength of a solution by the addition of a neutral salt (e.g. ammonium sulfate or sodium chloride) increases the hydrophobicity of protein molecules. This may be explained (somewhat simplistically) on the basis that the hydration of salt ions in solution results in an ordered shell of water molecules forming around each ion. This attracts water molecules away from protein molecules, which in turn helps to unmask hydrophobic domains on the surface of the protein. 

The staphylokinase gene has been cloned in E. coli, as well as various other recombinant systems. The protein is expressed intracellularly in E. coli at high levels, representing 10-15 per cent of total cellular protein. It can be purified directly from the clarified cellular homogenate by a combination of ion-exchange and hydrophobic interaction chromatography. 

Further characterization of the dendrimer-coupled protein complexes was studied by hydrophobic interaction chromatography carried out by purification over an octyl-Sepharose column. The products obtained by reaction of SIAB with ALP and its complex with third-generation dendrimer, ALP-E3, were... 

Since the hydrophobicity of styrene- or alkyl methacrylate-based monolithic matrices is too high to make them useful for hydrophobic interaction chromatography, porous monoliths based on highly hydrophilic copolymers of acrylamide and methylenebisacrylamide were developed [70,135]. The hydrophobicity of the matrix required for the successful separations of proteins is controlled by the addition of butyl methacrylate to the polymerization mixture. The suitability of this rigid hydrophilic monolith for the separation of protein mixtures is demonstrated in Fig. 21, which shows the rapid separation of five proteins in less than 3 min using a steeply decreasing concentration gradient of ammonium sulfate. 

Hydrophobic interaction chromatography is a form of reverse-phase chromatography most appropriate foi protein separations. 

M.T.W. Hearn, Reversed-phase and hydrophobic interaction chromatography of proteins and peptides, in HPLC of Biological Macromolecules, 2nd ed., K.M. Gooding and F. Regnier (Eds.), Marcel Dekker, New York, 2002, pp. 172-173. 

Practical Strategies for Protein Contaminant Detection by High-Performance Hydrophobic Interaction Chromatography... 

Hydrophobic interaction chromatography (HIC) occupies a unique niche in the field of analytical chromatography. A particular advantage of HIC is its unique selectivity. Whereas ion-exchange chromatography (IEC) principally reveals differences based on the surface charge of native proteins, HIC reveals differences based principally on their surface hydrophobicity. HIC is complementary to reversed-phase chromatography (RPC) in a different sense. Whereas HIC discriminates primarily on the basis of surface hydrophobicity, RPC principally reveals differences based on total hydrophobicity of all the hydrophobic residues of denatured proteins. 

Module 3, Column and Mobile Phase Design (CMP). This is the core module for ECAT. It can currently specify i) analytical column and mobile phase constituents for reverse phase chromatography of common classes of organic molecules ii) reverse phase, ion exchange phase and hydrophobic interaction chromatography of proteins and peptides iii) a limited set of specialty classes of molecules best treated by straight phase chromatography (e.g., mono- and disaccharides). The rules for selection of the HPLC detector are under development within Module 3. Some of the rules for detector mobile phase compatibility are already encoded. A set of rules for detector selection is ready but not yet encoded. 

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