Purpose To investigate human soluble TRAIL(sTRAIL)protein expression and purification and its potential anti-tumor activity on hepatocellular carcinoma(HepG2).Methods Soluble TRAIL gene ligated with expression vector pPIC9 was transfected into GS115(his4)and the recombination strain expressing sTRAIL was screened by MD plate.The effects of different media,methanol inducement period,methanol concentration,and pH were investigated and optimized using shaking flask.The anti-tumor activity of sTRAIL with HepG2 cells Was analyzed after purification.Results The highest expression of sTRAIL was obtained at pH 6.0,1% methanol in BMMY medium,with the concentration of(58.7±2.4)mg/L at 48 h.Recombinant sTRAIL protein could induce HepG2 cells apoptosis and inhibit HepG2 cells proliferation effectively.Conclusion The optimized condition of human sTRAIL expression and purification Was developed and the obtained recombinant sTRAIL protein may be a promising therapeutic agent for hepatocellular carcinoma.

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
Antioxidants ; pharmacology ; DNA, Complementary ; Electroporation ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics

Objective : To investigate the role of squalene epoxidase (SQLE) knockout in anti-tumor effect vial im- proving CD8 + T cell infiltration in melanoma tumor microenvironment． Methods : Both immunodeficient and immu- nocompetent mice were inoculated with SQLE knockout B16F10 cells to determine the cell-autonomous and non-au- tonomous regulation of malignancy．Antibody blockade ，Luminex multiplex assays ，and flow cytometry were em- ployed to explore the impact of SQLE gene knockout on the secretion of cytokines / chemokines and immune cell in- filtration．Bioinformatics analysis was conducted to validate the correlation between SQLE expression and immune infiltration as well as clinical prognosis in melanoma patients. Results : Compared with immunodeficient mice， SQLE knockout significantly inhibited melanoma proliferation in immunocompetent mice and prolonged their surviv- al．SQLE knockout induced the secretion of cytokines and chemokines from tumor cells，improved CD8 + T cell in- filtration in the tumor microenvironment，thereby potentiating anti-tumor immunity．Bioinformatics analysis sugges- ted a significant correlation between SQLE and its corresponding immune infiltration markers with the prognosis of melanoma patients． Conclusion SQLE regulates anti-tumor immunity by controlling cytokines and chemokines re- leasing in tumor microenvironment，thus holding promise as a novel tumor immunotherapy target and efficacy predic- tion molecular indicator.