Journal: PLoS ONE
Article Title: Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins
Figure Lengend Snippet: Expression and characterization of TTC-based fusions proteins. (A) The TTC DNA sequence was transferred to the pMS vector system using the SfiI/NotI sites to generate pMS-EGrB(R201K)-TTC and pMS-TTC-MAPTau. Abbreviations: CMV = cytomegalovirus promoter, Ig kappa = murine signal sequence for protein secretion into the cell culture supernatant, ECS = enterokinase cleavage site, His 10 = polyhistidine tag, IRES = internal ribosome entry site for the co-expression of eGFP, eGFP = enhanced green fluorescent protein. (B) EGrB(R201K)-TTC and TTC-MAPTau were expressed in HEK 293T cells and purified by IMAC. The GrB-(R210K)-TTC and TTC-MAPTau proteins were separated by denaturing SDS-PAGE followed by staining with Coomassie Brilliant Blue (left). Western blot analysis (right) using anti-polyhistidine and goat anti-mouse IgG (Fc specific) antibodies revealed protein bands of the anticipated sizes for GrBR201K-TTC (80 kDa) and TTC-MAPTau (93 kDa). Lane 1—Color Prestained Protein Standard, Broad Range (11–245 k); lane 2—GrBR201K-TTC, lane 3—TTC-MAPTau. (C) EGrB(R201K)-TTC was digested with enterokinase and granzyme B activity was tested using substrate Ac-IETD- p NA (white circle) compared to uncleaved EGrB(R201K)-TTC (black triangle) and the mock-protein (white triangle). The enzymatic activity of the granzyme B domain was determined using a colorimetric assay and the absorbance at 405 nm was monitored for 60 min in 2-min intervals. (D) An XTT-based cell viability assay was carried out using serial dilutions of the novel TTC-fusion proteins against the mouse TTC-reactive hybridoma cell line 5E4 (72 h, 37°C, 5% CO 2 ). The data are means ± standard deviation (SD) of technical triplicates of three independent experiments (n = 3).
Article Snippet: Purified proteins were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining against Color Prestained Protein Standards, Broad Range (New England Biolabs, Ipswich, Massachusetts).
Techniques: Expressing, Sequencing, Plasmid Preparation, Cell Culture, Purification, SDS Page, Staining, Western Blot, Activity Assay, Colorimetric Assay, Viability Assay, Standard Deviation