Expression, Purification, And Characterization Of Recombinant Human Soluble BAFF

Part I: Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastorisHuman B lymphocyte stimulator (BAFF), a new member of tumor necrosis factor (TNF) superfamily, stimulates proliferation of B lymphocyte and immunoglobulin in the presence of anti-IgM. BAFF plays an important role in the regulation of humoral immunity. Therefore, BAFF, or its antagagonists can be developed as protein drug targeted to immune deficiencies disorders or autoimmune diseases. The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102mg. protein was obtained in high purity from 1L of the supernatant and its identity to hsBAFF was confirmed by NH2-terminal amino acid sequence analysis. Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggested that the P. pastoris expression system could be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.Part II: Baculovirus-mediated HCC targeted gene therapy with the hTERT promoter and poptinReports have described that baculoviruses can transduce a broad spectrum of primary and established mammalian cells, especially, hepatocellular cell which shows the baculoviruses could serve as a new gene transfer vehicle for HCC gene therapy. In this report, we investigated the modification of baculovirus vector and the way to deliver exogenous gene into HepG2 cells. On the base of Bac-to-Bac baculovirus insect cell expression system, two recombinant baculoviruses (BacV-CMV-EGFP, BacV-hTERT-EGFP) were constructed containing CMV and hTERT promoter which controll the expression of a reporter gene (EGFP). We found that CMV promoter could direct expression of reporter gene in Sf9 cells, and expressed EGFP more effiecient than hTERT promoter in HepG2 cells. Furthermore, we put the apoptin under the control of the hTERT promoter and constructed recombinant baculoviruses BacV-hTERT-Apoptin. The result showed that this recombinant baculoviruses BacV-hTERT-Apoptin could induce HepG2 cells apoptosis. So we can draw a conclusion that recombinant baculoviruses BacV-hTERT-Apoptin could serve as a convenient and high-performance carrier for targeted gene therapy of HCC.