Study on Expression Condition Optimization of Two Recombinant Methioninases and Their Inhibitory Effects on Human Lung Adenocarcinoma Cells GLC / 中国药房

ABSTRACT

OBJECTIVE： To optimize the expression induction condition of two recombinant methioninases， and to investigate their inhibitory effects on the proliferation of human lung adenocarcinoma cells GLC. METHODS： Recombinant methioninases expression plasmid PGEX-4T1-4A1-MGL and PGEX-4T1-3B8-MGL were transfected into competent Escherichia coli Dh5α， and induced by isopropyl-β-D-thiogalactoside. Using the expression level of target protein as index， the initial OD600 nm value before induction， culture temperature and induction time were optimized by single factor test. The recombinant methioninase 4A1-MGL and 3B8-MGL were purified by affinity chromatography. The concentration of recombinant methioninase was detected by Coomassie blue method. The purity of the product was detected by sodium lauryl benzene sulfonate-polyacrylamide gel electrophoresis； its activity was detected by spectrophotometry. The proliferation of cells was detected by MTT assay after treated with low-dose， medium-dose and high-dose of recombinant methioninases （4A1-MGL or 3B8-MGL was 0.1， 0.2， 0.4 U/mL） for 24， 48， 74 h. Inhibitory rate of cells were calculated. RESULTS： The optimal induction condition of two recombinant methioninases included that initial OD600 nm of 0.9， culture temperature of 37 ℃， induction time of 5 h. The results of validation test showed that protein expression level of 4A1-MGL was 1.52±0.04， that of 3B8-MGL was 1.28±0.03 （RSD＜3％，n＝3）. After purification， the concentration， purity and activity of 4A1-MGL were （0.70±0.02）mg/mL， （96.42±3.15）％ and （0.45±0.02）    U/mg； and those of 3B8-MGL were （0.56±0.02）mg/mL， （97.43±2.96）％ and （0.91±0.03）U/mg. After treated with low-dose and medium-dose of 4A1-MGL and 3B8-MGL for 48 and 72 h， treated with high-dose of 4A1-MGL and 3B8-MGL for 24， 48 and 72 h， inhibitory rate of GLC cell was increased significantly， and high-dose group for 72 h was significantly higher than low-dose and medium-dose groups at same time point （P＜0.05）. CONCLUSIONS： The induction conditions of recombinant methioninase expression are successfully optimized in this study. The obtained 4A1-MGL and 3B8-MGL could inhibit the proliferation of GLC cells in a dose-dependent manner.