Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

Abstract

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their non-prenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography media that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target (“CAAX box”) sequences were enzymatically prenylated in vitro with natural and non-natural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction—farnesylation of a mCherry-CAAX fusion construct—was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a non-natural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate, as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.

Solid-State Nanostructured Materials from Self-Assembly of a Globular Protein–Polymer Diblock Copolymer

“…The band at 28,000 g/mol corresponds to unconjugated mCherry, while the bands at 19,000 g/mol and 9,000 g/mol correspond to the partial hydrolysis of the mCherry chromophore acylimine bond during SDS-PAGE analysis. 50–53 Lane analysis of the crude reaction mixture revealed a conversion of approximately 78%, comparable to previously reported conversions for thiol-maleimide couplings to globular proteins. 54–56 Although the eight-fold excess of PNIPAM used gave maximum conversion of the mCherryS131C, decreasing to a five-fold excess still yielded approximately 70% conversion.…”

“…50 The native gel (Supporting Information), run at a high protein concentration, shows only a single prominent protein band corresponding to the mCherry and a higher molecular weight band corresponding to dimerization through the formation of a disulfide bond. Consistent with previously reported expressions of mCherry, 53, 66 it is noted that the lower molar mass fragments are not observed by SDS-PAGE in more dilute samples. The observation of a single dominant band in the native gel indicates that cleavage occurs only during SDS-PAGE sample preparation.…”

Simultaneous ligand fishing and identification of human serum albumin binders from Eucommia ulmoides bark using surface plasmon resonance-high performance liquid chromatography–tandem mass spectrometry

“…The flavonoids and phenolic acids, quercetin-3-O-sambubioside (14), rutin (16), isoquercitrin (18), 3-(3,4-dihydroxyphenyl)propionic acid (4), protocatechuic acid (6), chlorogenic acid (7) as well as vanillic acid (12) were identified by comparison of their t R , UV and MS/MS with those of reference standards. These compounds were frequently found in natural products, and were also previously isolated and identified from E. ulmoides [4][5][6].…”

“…Up to now, many ligand fishing methods combined with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) have been employed, which include, but are not limited to, centrifugation [8], ultrafiltration [6,9,10], equilibrium dialysis [11], microdialysis [12], magnetic beads [13], affinity chromatography [14]. All of the methods combined bioactivity assay and analysis power to accommodate simultaneous readouts of biochemical fingerprint and unequivocal structural information of bioactive compounds [15].…”

Protein Purification by Affinity Chromatography

“…The ligand molecule to be used should contain a group capable of being chemically modified, often an amino group, which will allow connection with the matrix without destroying its capacity to bind to the molecule of interest. One example is the agarose, a polysaccharide obtained from agar, which provides numerous free hydroxyl groups and is the most widely used (Chung et al, 2009). The ligand may be covalently bound to it through a two step process.…”