T-helper-associated cytokines expression by peripheral blood mononuclear cells in patients with polypoidal choroidal vasculopathy and age-related macular degeneration.

BACKGROUND: Immune responses play a key role in the pathogenesis and progression of polypoidal choroidal vasculopath (PCV) and age-related macular degeneration (AMD). In this study, we determined the Th cell-associated immune responses by measuring the cytokine expression of peripheral blood mononuclear cells (PBMC) in both PCV and neovascular AMD (nAMD) patients. METHODS: Twenty-seven nAMD patients, 33 PCV patients and a gender- and age-matched group of 18 healthy individuals were involved in this study. The Th-cell cytokine profiles including levels of interferon-gamma (INF-γ), interleukin (IL)-17A and IL-4 in cultures of PBMCs were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: IFN-γ,IL-17A and IL-4 production was significantly increased after stimulation with PHA. The levels of IFN-γ and IL-4 in PHA-stimulated cultures were higher in PCV and nAMD patients than that in healthy controls (P = 0.038,P = 0.014), while no difference was found between PCV and nAMD (all P > 0.05). No significant difference in IL-17A level in PHA-stimulated cultures was found among PCV, nAMD and control groups (P > 0.05). CONCLUSIONS: These findings suggest that circulating IFN-γ and IL-4 producing Th1 and Th2 cells may involve in the pathogenesis of PCV and nAMD. PCV may have the similar immune responses with nAMD.

Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its "oxidizing" mutant.

AIM: To study the influence of redox environment of Escherichia coli (E. coli) cytoplasm on disulfide bond formation of recombinant proteins. METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF, and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3) and M15(pREP4) respectively. At the same time, both plasmids were transformed into a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively. RESULTS: All recombinant E. coli strains could efficiently produce target proteins. The level of BbFGF in BL21(DE3) was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm, and all the target proteins became soluble in Origami(DE3). The bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED(50) of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 microg/L and 2.2 microg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15(pQE-HBscFv) or Origami(pQE-HBscFv). But the supernatant of Origami(pQE-HBscFv) lysate displayed weak bioactivity and its counterpart from M15(pQE-HBscFv) did not display any bioactivity. The soluble HBscFv in Origami(pQE-HBscFv) was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62 x 10(7) mol/L. The yield of native HBscFv refolded from inclusion body in M15(pQE-HBscFv) was 30-35 mg/L and the affinity constant was 1.98 x 10(7) mol/L. There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains. CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.

[Expression of a human single-chain Fv antibody against HBsAg in Pichia pastoris].

To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.

[Influence of the reductase deficient Escherichia coli on the solubility of recombinant proteins produced in it].

The cytoplasm of E. coli is a reducing environment where cysteines do not engage in disulfide bonds. Any disulfide bonds that do appear are rapidly reduced through the action of disulfide reducing enzymes such as thioredoxin and glutaredoxin. To study the influence of E. coli cytoplasm on the solubility of recombinant proteins produced in it, bovine fibroblast growth factor (BbFGF), with single disulfide bond, and anti-HBsAg single-chain Fv (HBscFv), with two disulfide bonds, were selected as the pattern molecules of simple protein and complex protein, respectively. pJN98-BbFGF, a BbFGF expressing plasmid based on the vector pET3c, was constructed and transformed into normal host BL21(DE3) and a reductase deficient strain, E. coli Origami(DE3). At the same time, pQE-HBscFv, a HBscFv expressing plasmid was constructed and transformed into M15 [pREP4] and Origami(DE3). The recombinant BbFGF and HBscFv were produced in 2 types of bacteria and their solubilities and bioactivities were determined, respectively. It was found that the majority of BbFGF had formed inclusion body in the cytoplasm of BL21 (DE3) and all of them turned into soluble protein in Origami(DE3). It was also found the productivity of BbFGF in Origami (DE3) was 5% - 10% of the total protein and the value was 15% - 23% in BL21(DE3). BbFGFs produced in 2 recombinant bacteria were purified by cation exchange and heparin affinity chromatography. MTT assay revealed that the bioactivity of BbFGF purified from Origami(DE3) was higher than its counterpart from BL21(DE3). The ED50 of BbFGFs from different bacteria was 1.6ng/mL and 2.2ng/mL, respectively. As far as HBscFvs, both of them formed inclusion body in the cytoplasm of M15 [pQE-HBscFv] and Origami [pQE-HBscFv]. The inclusion body was solubilized in 6mol/L GuHCl, purified with a His-Trap column and then refolded by dialysis step-by-step against buffers containing downtrend concentration of GuHCl. Indirect ELISA was applied to determine the HBsAg binding activity of HBscFvs. It was found there was no obvious difference between the bioactivity of refolded HBscFvs produced from 2 recombinant bacteria. On the other hand, the supernatant of Origami [pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15 [pQE-HBscFv] displayed without any bioactivity. The soluble HBsFv in the cytoplasm of Origami [pQE-HBscFv] was purified by cation exchange and immobilized metal affinity chromatography (IMAC) and the yield was 1 - 2mg/L. Those results suggested that modification of the redox environment of E. coli cytoplasm greatly improved the solubility of recombinant disulfide-bonded proteins produced in it. In the next step, we had like to co-express of molecular chaperones or refoldase to raise the yield of soluble recombinant proteins, as well as optimizing the culture condition of the "oxidizing" E. coli.