PubMed · 15669252
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Ryoichi Arai, Mikako Shirouzu, Shigeyuki Yokoyama, Hiroshi Hirota. 2004. [Tagged-MS method].. https://pubmed.ncbi.nlm.nih.gov/15669252/
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Green fluorescent protein (GFP) has been proposed as an ideal choice for a protein-based biological indicator for use in the validation of decontamination or disinfection treatments. In this article, we present a potentially scalable and cost-effective way to purify recombinant GFP, produced by fermentation in Escherichia coli, by affinity-enhanced extraction in a two-phase aqueous micellar system. Affinity-enhanced partitioning, which improves the specificity and yield of the target protein by specific bioaffinity interactions, has been demonstrated. A novel affinity tag, family 9 carbohydrate-binding module (CBM9) is fused to GFP, and the resulting fusion protein is affinity-extracted in a decyl beta-D-glucopyranoside (C10G1) two-phase aqueous micellar system. In this system, C10G1 acts as phase forming and as affinity surfactant. We will further demonstrate the implementation of this concept to attain partial recovery of affinity-tagged GFP from a clarified E. coli cell lysate, including the simultaneous removal of other contaminating proteins. The cell lysate was partitioned at three levels of dilution (5x, 10x, and 40x). Irrespective of the dilution level, CBM9-GFP was found to partition preferentially to the micelle-rich phase, with the same partition coefficient value as that found in the absence of the cell lysate. The host cell proteins from the cell lysate were found to partition preferentially to the micelle-poor phase, where they experience less excluded-volume interactions. The demonstration of proof-of-principle of the direct affinity-enhanced extraction of CBM9-GFP from the cell lysate represents an important first step towards developing a cost-effective separation method for GFP, and more generally, for other proteins of interest.
Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.
Lipoxygenases are lipid-peroxidizing enzymes, which have been implicated in the pathogenesis of important diseases. They consist of a single polypeptide chain, which is folded into a two-domain structure. The large catalytic domain contains the putative substrate-binding pocket and the catalytic non-heme iron. To identify structural elements of the rabbit 12/15-lipoxygenase that are involved in enzyme/substrate and/or enzyme/product interaction, we synthesized a set of radioactively labeled lipoxygenase substrates carrying a photoreactive azido group (17-azido-ETE, 18-azido-ETE, 19-azido-ETE) and used these compounds as affinity probes. After photoaffinity labeling, the enzyme was digested proteolytically and modified tryptic cleavage peptides were identified by a combination of radio-HPLC and mass spectral analysis. Following this strategy, we observed covalent linkage of a cleavage peptide that contained Ile593, which has previously been identified as the sequence determinant for the positional specificity. These data are consistent with the previous suggestion that this peptide lines the substrate-binding pocket. Surprisingly, we also observed strong labeling of cleavage peptides originating from the N-terminal beta-barrel domain, and our mass spectral data suggested covalent linkage of oxidized affinity probes. Taken together, these results confirm the previous conclusion that Ile593 and surrounding amino acids are constituents of the active site, but they also implicate the N-terminal beta-barrel in enzyme/substrate and/or enzyme/product interaction.