Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis

Objective To investigate the role and mechanism of nico-tinamide adenine dinucleotide phosphate oxidase 4 (NAPHD oxidase 4,Nox4)-mediated reactive oxygen species (ROS) generation on high-dose dexamethasone (DEX) induced apoptosis in osteoblasts.Methods According to culture conditions,3rd passage of murine osteoblastic MC3T3-E 1 cells were divided into control group,Dexamethasone group,Dexamethasone+NAC (N-acetyl-L-cysteine) group,NAC group,Dexamethasone+DPI (Diphenyleneiodonium) group and DPI group.24 hours after culture,the morphology of osteoblasts was observed by inverted phase contrast microscope.Cell viability was determined by MTT assay.The generation of ROS in osteoblasts was measured using a fluorescent probe DCFH-DA.The apoptosis of each group was observed through Hoechst staining.The mRNA level and protein expression of Nox4 were detected by real-time quantitative PCR and Western Blot.In addition,after silence of Nox4 with small interfering RNA (siRNA),the ROS generation was further detected by a fluorescent probe DCFH-DA.Results After treatment with 1000 nmol DEX for 24 hours,compared to control group,the results of inverted phase contrast microscope and MTT showed that osteoblasts in DEX group exhibited more obvious signs of shrinkage and deformation with decreased cell viability.After intervene with NAC and DPI,morphology of osteoblasts was good with increased viability of osteoblasts.Compared to control group (5.86％± 0.28％),the production of ROS in DEX group (45.14％±1.49％) was significantly increased (P=0.000).The apoptotic rates in DEX group (29.60％± 1.52％) was significantly increased compared with control group (4.12％±0.67％) (P=0.000).Compared to DEX group,the production of ROS generation in DEX+NAC group (28.06％±1.61％) and DEX+DPI group (23.70％±1.28％) was significantly decreased (P=0.000).It presented that NAC or DPI significantly decreased the formation of ROS.Compared to DEX group,the apoptotic rate in DEX+NAC group (8.94％± 1.47％) and DEX+DPI group (12.96％±2.03％) was significantly decreased (P=0.000).It presented that NAC or DPI significantly decreased osteoblast apoptosis.In addition,the Nox4 mRNA level in DEX group was 2.67-fold compared with control group (t=-10.301,P=0.009).The difference had statistically significance.The protein expression of Nox4 in DEX group was 2.37-fold compared with control group (t=-15.542,P=0.004).The difference has statistically significance.After silence of Nox4 by siRNA,the generation of ROS in DEX+Nox4 siRNA group (14.53％± 1.00％) was decreased by 16.92％ compared with DEX group 31.45％±0.72％ (P=0.000).The difference had statistically significance.Conclusion Nox4-mediated ROS generation plays an important role in osteoblasts apoptosis induced by high-dose dexamethasone.It provided us the new target in the management of Nox4 to provide possible therapy for steroid-induced avascular necrosis of the femoral head (SANFH).