Structure, catalytic mechanism, and evolution of the glutathione transferases.

http://home.sandiego.edu/~josephprovost/Making%20the%20most%20of%20affinity%20tags.pdf

Making the most of affinity tags.

PROBLEM TO BE SOLVED: To perform molecular weight measurement and separation of a fluorine containing organic substance slightly soluble in a general organic solvent by means of size exclusion chromatography by using a solvent containing dichloro pentafluoropropane as a mobile phase and using polymethyl methacrylate with a known molecular weight as a molecular weight measurement standard specimen. SOLUTION: As a mobile phase. a solvent containing HCFC225 such as a solvent containing only HCFC225 and a mixture solvent containing HCFC225 and other solvents are available. HCFC225 is selected from one or more kinds of substances selected from chlorinated fluorinated hydrocarbons with a molecular formula represented by C3 HF3 Cl2 . As the HCFC225, several kinds of isomers with the same molecular formula and different structures are known, however, HCFC225ca (CF3 CF2 CHCl2 ), HCFC225cb (CF2 ClCF2 CFHC1), and a mixture of them are desirable. As a molecular weight measurement standard specimen, polymethyl methacrylate with a known molecular weight is used.

https://www.springer.com/productFlyer_978-3-540-65635-7.pdf?SGWID=0-0-1297-1427978-0

Size Exclusion Chromatography

Glutathione transferases in the genomics era : New insights and perspectives

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/biot.201100155

Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications

A glutathione S-transferase (Sj26GST) from Schistosoma japonicum, which functions in the parasite's Phase II detoxification pathway, is expressed by the Pharmacia pGEX-2T plasmid and is used widely as a fusion-protein affinity tag. It contains all 217 residues of Sj26GST and an additional 9-residue peptide linker with a thrombin cleavage site at its C-terminus. Size-exclusion HPLC (SEC-HPLC) and SDS-PAGE studies indicate that purification of the homodimeric protein under nonreducing conditions results in the reversible formation of significant amounts of 160-kDa and larger aggregates without a loss in catalytic activity. The basis for oxidative aggregation can be ascribed to the high degree of exposure of the four cysteine residues per subunit. The conformational stability of the dimeric protein was studied by urea- and temperature-induced unfolding techniques. Fluorescence-spectroscopy, SEC-HPLC, urea- and temperature-gradient gel electrophoresis, differential scanning microcalorimetry, and enzyme activity were employed to monitor structural and functional changes. The unfolding data indicate the absence of thermodynamically stable intermediates and that the unfolding/refolding transition is a two-state process involving folded native dimer and unfolded monomer. The stability of the protein was found to be dependent on its concentration, with a delta G degree (H2O) = 26.0 +/- 1.7 kcal/mol. The strong relationship observed between the m-value and the size of the protein indicates that the amount of protein surface area exposed to solvent upon unfolding is the major structural determinant for the dependence of the protein's free energy of unfolding on urea concentration. Thermograms obtained by differential scanning microcalorimetry also fitted a two-state unfolding transition model with values of delta Cp = 7,440 J/mol per K, delta H = 950.4 kJ/mol, and delta S = 1,484 J/mol.

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

Solvent-induced unfolding of porcine class pi glutathione S-transferase (pGST P1-1), a homodimeric protein, was monitored under equilibrium conditions using different physicochemical parameters (tryptophan fluorescence, anisotropy, degree of tyrosine exposure, binding of 8-anilino-1-naphthalenesulphonic acid, size-exclusion HPLC). The coincidence of unfolding curves obtained with functional (enzyme activity) and structural probes (anisotropy), the absence of thermodynamically stable intermediates such as a folded monomer (determined by binding of 8-anilino-1-naphthalenesulphonic acid and size-exclusion HPLC), and the dependence of pGST P1-1 stability upon protein concentration (measured with structural and functional probes), indicate a cooperative and concerted two-state unfolding transition between native dimeric pGST P1-1 and unfolded monomeric enzyme.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1995.0614h.x

Native Dimer Stabilizes the Subunit Tertiary Structure of Porcine Class pi Glutathione S‐transferase

Porcine class π glutathione S-transferase: anionic ligand binding and conformational analysis

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2896%2900768-5

Julija Erhardt

Papers

Conformational stability of pGEX-expressed Schistosoma japonicum glutathione S-transferase: a detoxification enzyme and fusion-protein affinity tag.

Native Dimer Stabilizes the Subunit Tertiary Structure of Porcine Class pi Glutathione S‐transferase

Porcine class π glutathione S-transferase: anionic ligand binding and conformational analysis

Effect of glutathione, glutathione sulphonate and S‐hexylglutathione on the conformational stability of class pi glutathione S‐transferase