Figure 1.
A schematic genetic map of the sCD83 expression vector.
The sCD83 gene was inserted between the Xho I and EcoR I sites in pPIC9K and in-frame with the Kex2 cleavage site in the sequence of the α-factor secretion signal to create the expression vector pPIC9K-sCD83.
Figure 2.
The screening and optimization of sCD83 expression in flasks.
(A) The screening of high-expressing colonies of sCD83 from YPDG plates. 1–13: the colony number, Ctr. : the GS115/pPIC9K control. (B) The optimization of the induction temperature in flasks. (C) The optimization of the induction pH in flasks. Each experiment with these conditions was performed in triplicate.
Figure 3.
The fermentation of sCD83 in P. pastoris.
(A) The plotted parameters show the DO, the pH, the temperature, and the feeding speed during fermentation. (B) Methanol feeding and the concentration during fermentation. (C) The biomass increases in DCW, WCW, and OD600 during induction. (D) Western blot analysis of the time period for expression during fermentation.
Figure 4.
The purification and characterization of sCD83.
(A) The isolation of sCD83 by HisS from the fermentation supernatant. HisS: His-Select chromatography. (B) The further purification of sCD83 by S-200. S-200: size-exclusion chromatography S-200 HR, M: pre-stained protein molecular marker. (C) Approximately 20 µg of sCD83 was incubated with 2.0 IU of PNGase F according to the manufacturer’s instructions. The reaction mixture was analyzed in 15% SDS-PAGE and stained with Coomassie blue. Western blotting was performed using anti-CD83 polyclonal antibodies under reducing conditions.
Figure 5.
The specificity detection of sCD83.
sCD83 has the similar epitope as the membrane form. (A, B) FACS competition tests of sCD83. Mature DCs were coated with the indicated antibody for DC surface molecules, and the indicated concentration of sCD83 was added. Representative FACS histograms are shown. (B) The data are from (A) an experiment that is representative of at least 3 independent experiments. (C, D) FACS competition tests of an anti-sCD83 pAb. Mature DCs were coated with the indicated antibody for DC surface molecules, and the indicated concentration of sCD83 polyclonal antibody was added. Representative FACS histograms are shown. (D) The data are from (C) an experiment that is representative of at least 3 independent experiments. The means ± the standard error of the mean (SEM) of the MFI are shown (B, D).
Figure 6.
Biological activity determination of sCD83.
sCD83 bound to the putative CD83 receptor on monocytes but not on T cells. (A and B) PBMC were incubated with soluble CD83 or BSA, and then coated with either PE-anti-CD83 (A) or anti-His followed by FITC-Anti-mouse IgG (B). Cells were then coated with anti-CD3 or anti-CD14 to distinguish the monocyte and T cells. CD14+ monocytes and CD3+ T cells were gated and analysis for CD83 receptor expression. (C) sCD83 suppressed ConA-stimulated PBMC proliferation. PBMCs were stained with CFSE, stimulated with ConA, and cultured with or without 20 µg/mL of sCD83 for the indicated time periods. (D) The means ± the standard error of the mean (SEM) of the MFI from (C) are shown as bar graphs. (E) sCD83 activated the NF-κB pathway. Isolated human PBMCs stimulated by sCD83 (10 µg/mL) were cultured in RPMI 1640 medium that contained 10% fetal bovine sera at 37°C for 12 h. The PBMCs were collected and lysed to detect the activation of the NF-κB pathway using Western blotting.