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

Abstract

BackgroundHydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported.Methods and ResultsBacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation.ConclusionsConducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in development other of HIC-compatible protein purification schemes.

Economic analysis of the production and recovery of green fluorescent protein using ATPS-based bioprocesses

“…Therefore, the purification of pharmaceutical biocompounds is usually complex and based in multi-step platforms [22]. For example, the current strategies for GFP purification involve, mainly, the use of multiple chromatographic units, which are complex and costly [18,[23][24][25][26][27].…”

Extraction of recombinant proteins from Escherichia coli by cell disruption with aqueous solutions of surface‐active compounds

“…4 The GFP has been recognized for its high stability towards denaturing conditions/compounds, such as temperature, pH, proteases, organic solvents, detergents, or chaotropic agents like urea up to 8 mol L -1 . 3,5 In addition, GFP has a unique spontaneous and natural emission of green light fluorescence when excited by UV light, 6 which is useful in real-time imaging applications. However, since GFP is produced intracellularly in E. coli, an additional cell disrupting stage is required.…”

Automated Hydrophobic Interaction Chromatography Screening Combined with In Silico Optimization as a Framework for Nondenaturing Analysis and Purification of Biopharmaceuticals

“…Automation has been instrumental in achieving fast method development that can be used to monitor the purity, stability, and in-depth characterization of biologics. While numerous multicolumn/multieluent separation platforms have been already reported for IEX, RPLC, and multidimensional LC, − automated HIC screening frameworks remain an underdeveloped area with currently available setups that do not integrate automated salt screening and are mainly dedicated to protein purification. , Herein, a multicolumn and multieluent separation platform for HIC method scouting, optimization, and analytical-scale purification is introduced. Columns with complementary selectivity and various eluent salts were investigated and deployed to enable a wide range of biopharmaceutical applications including unconjugated proteins, mAbs, and ADC analysis.…”