title: Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences.
abstract: Hydrophobic interaction chromatography (HIC) has been developed as a powerful technique for separating and purifying proteins. In this study, we have characterized the ability of new multimodal pH-HIC media to resolve proteins with only small differences in their primary structures. This was done by determining the retention times of different green fluorescent protein (GFP) mutants prepared from Escherichia coli extracts. The mutants, modified with single or double hydrophobic amino acid substitutions in two positions, N212 and T230, could be resolved successfully, up to 2.1 column volumes in retention difference for single substitutions and 2.6 column volumes for double substitutions, at two pH and on two media with varying ligand density. The retention times also correlated well with calculated theoretical retentions (R2=0.91) using a hydrophobic descriptor. This medium can therefore be very useful in a final polishing step during purification and the protein library prepared represents a good screening set in validating and characterizing new future media due to the accessible, but yet, extremely small differences in protein structure.
authors.affiliation: Department of Pure and Applied Biochemistry, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-211 00, Lund, Sweden.
authors.forename: Kristian
authors.initials: K
authors.lastname: Becker
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Hallgren
authors.forename: Enrique
authors.initials: E
authors.lastname: Carredano
authors.forename: Ronnie
authors.initials: R
authors.lastname: Palmgren
authors.forename: Leif
authors.initials: L
authors.lastname: Bülow
mesh_terms: D019943:Amino Acid Substitution; D002853:Chromatography, Liquid / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D011487:Protein Conformation
chemical_list: D049452:Green Fluorescent Proteins