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Fig. 6 | Microbial Cell Factories

Fig. 6

From: SHTXTHHly, an extracellular secretion platform for the preparation of bioactive peptides and proteins in Escherichia coli

Fig. 6

Detection of the cellular outputs mediated by the SHTXTHHly-produced mFc-based antibodies. A Schematic diagram of the SHTXTHHly-produced mFc-based antibodies. TEV cs: TEV enzyme cleavage site; H’: Mutated hinge; mFc: Monomeric Fc; Hly218: the last 218 amino acids of hemolysin A. The site mutations were indicted by red points. B SDS-PAGE analysis of the purification of RGDSH’mFc. W: Whole-cell lysate; S: Supernatant; FT: Flow-through; E: Eluent; T: TEV enzyme digestion production of the eluent; 1: First round nickel affinity chromatography; 2: Second round nickel affinity chromatography. C Schematic diagram of the binding between CD64 chimeric Jurkat cells and the SHTRGDSH’mFcTHHly fusion protein. The red squares indicate the downstream activation elements, namely CD28, CD137 (41BB), and CD247 (CD3ζ). To measure the binding affinities, JK-64CAR cells (2E5) were incubated with target proteins (7 nM) and the second antibody in sequence, and then cell staining was measured by FACS (D). U87 MG cells, CFSE-stained JK-64CAR cells, and mFc-based antibodies were incubated for 24 h, then cell activation (E) and IL-2 release (F) were measured by FACS and the ELISA kit, respectively. The 1-tailed Student's t-test was used to determine statistical differences between the two groups

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