Hgstp1-1 And Its Cysteine ​​mutants Of The Prokaryotic Expression Vector, Induced Expression, Purification And Partial Characterization Of Two Kinds Of Egfp Was-hstrail Fusion Protein Expression Vector And Fusion Protein Purification And Analy

The bacterial expression and purification of human glutathione S-transferase P1-1(hGST P1-1), as a hexahistidine-tagged polypeptide, was performed. Site-directed mutagenesis was used to construct mutants in which alanine replaced two (C47A/C101A), three (C14A/C47A/C101A) or all four (C14A/C47A/ C101A/C169A) cysteine residues using the plasmid for the wild type enzyme. Analysis of their catalytic activities and kinetic parameters suggested that cysteins are not essential for the catalytic activity but may contribute to some extent to the catalytic efficiency. Moreover, on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions, hexahistidine-tagged hGST P1-1 (His₆-hGST P1-1) treated with 1 mM H₂O₂ showed at least three extra bands, in addition to the native His₆-hGST P1-1 subunit band. These extra bands were not detected in the cysteinyl mutants. Thus, it indicated that disulfide bonds were formed mainly within subunits between cysteine residues, causing an apparent reduction in molecular weight, only small amounts of binding between subunits being observed. The extracellular portion (amino acids 95-281 or 114-281) of the human TNF-related apoptosis-inducing ligand (sTRAIL) was genetically linked to the C-terminus of the fluoresce-enhanced green fluorescent protein variant (EGFP) to generate two versions of EGFP-sTRAIL fusion protein, designated EGFP-sTR95 and EGFP-sTR114 respectively. The two versions of EGFP-sTRAIL fusion protein both induce extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. In addition, the two versions of fusion protein retain similar fluorescence spectra to those of EGFP, and have shown the specific binding to TRAIL receptor positive-cells, thus the stained cells could be analyzed with flow cytometry. Hence, the two versions of fusion protein represent a readily obtainable source of biologically active sTRAIL that may prove useful in exploit fully the characteristics of both the soluble TRAIL and its receptor system.