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Protocol for the purification of protected carbohydrates: toward coupling automated synthesis to alternate-pump recycling high-performance liquid chromatography.

ABSTRACT: Given recent advances in automated oligosaccharide synthesis, analytical techniques that can be coupled to a synthetic framework are needed to not just identify but also purify to homogeneity protected carbohydrate compounds at levels of ?99.5% purity. Herein, an alternate-pump recycling high-performance liquid chromatography (R-HPLC) method has been developed to allow purification of protected carbohydrates at levels of ?99.5% purity.

SUBMITTER: Nagy G

PROVIDER: S-EPMC5123635 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Protocol for the purification of protected carbohydrates: toward coupling automated synthesis to alternate-pump recycling high-performance liquid chromatography.

Nagy Gabe G Peng Tianyuan T Kabotso Daniel E K DE Novotny Milos V MV Pohl Nicola L B NL

Chemical communications (Cambridge, England) 20161101 90

Given recent advances in automated oligosaccharide synthesis, analytical techniques that can be coupled to a synthetic framework are needed to not just identify but also purify to homogeneity protected carbohydrate compounds at levels of ≥99.5% purity. Herein, an alternate-pump recycling high-performance liquid chromatography (R-HPLC) method has been developed to allow purification of protected carbohydrates at levels of ≥99.5% purity. ...[more]

PMID: 27775116

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Project description: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.

Dataset Information

Protocol for the purification of protected carbohydrates: toward coupling automated synthesis to alternate-pump recycling high-performance liquid chromatography.

Publications

Protocol for the purification of protected carbohydrates: toward coupling automated synthesis to alternate-pump recycling high-performance liquid chromatography.

Similar Datasets

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