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.

High throughput screening of mixed-mode sorbents and optimisation using pre-packed lab-scale columns for the purification of the recombinant allergen rBet v 1a

“…The mixed-mode sorbents investigated here proved to offer new selectivities inherent to the multi-modal mechanism involved at binding and desorption. This has been also observed recently on new multimodal pH-HIC sorbents using green fluorescent protein mutants modified with single or double amino acid substitutions [21].…”

Plasmid DNA purification using a multimodal chromatography resin

“…Consequently, supports with larger pore sizes (Tarmann and Jungbauer, 2008), monoliths (Yamamoto et al, 2007(Yamamoto et al, , 2009), use of different ligand densities (Chen et al, 2011), and membranes (Zhong et al, 2011) have all been examined to promote higher pDNA yields. Multimodal chromatography (MMC) has been developed into a versatile and general purification approach and has frequently been used for protein and antibody purifications (Becker et al, 2008;Chung et al, 2010;Freed et al, 2011;Hou and Cramer, 2011;Kallberg et al, 2012). This modality utilizes more than one form of physical interaction between the stationary phase and the target molecules, for example, ion-exchange and HIC (Becker et al, 2008;Chung et al, 2010;Hou and Cramer, 2011) or reversed-phase and hydrophilic interactions (Lammerhofer et al, 2008;Wu et al, 2008).…”

“…Multimodal chromatography (MMC) has been developed into a versatile and general purification approach and has frequently been used for protein and antibody purifications (Becker et al, 2008;Chung et al, 2010;Freed et al, 2011;Hou and Cramer, 2011;Kallberg et al, 2012). This modality utilizes more than one form of physical interaction between the stationary phase and the target molecules, for example, ion-exchange and HIC (Becker et al, 2008;Chung et al, 2010;Hou and Cramer, 2011) or reversed-phase and hydrophilic interactions (Lammerhofer et al, 2008;Wu et al, 2008). Compared with the traditional mono-modal ligands, a higher selectivity can often be achieved with these ligands.…”

Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

“…Examples of previously reported GFP purification approaches include affinity purification with monoclonal antibodies [13], sequential HPLC-based SEC-IEC [14], preparative PAGE [15], and HIC [16], [17], [18]. While these previously described techniques result in highly purified GFP, each contains elements that may limit its adaptability to other HIC-compatible protein purifications.…”

“…While these previously described techniques result in highly purified GFP, each contains elements that may limit its adaptability to other HIC-compatible protein purifications. Examples of potential limitations of these previously described methods include exposure to organic solvents (e.g., three-phase partitioning [18]), heat (e.g., 60–72°C incubation [16], [17]), electro-elution [15], high pressure [14], and GFP antigen recognition [13]. There are few examples in the literature providing direct comparisons of untagged GFP purification elution profiles or eluate analyses following HIC column scouting.…”