科研之友
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摘要
BACKGROUND: TNF-alpha is a pleiotropic cytokine that plays an important role in both physiological and pathological conditions. An increasing demand of large amount of bioactive TNF for use in clinic and drug development strategies make this cytokine as one of the most important biotechnologically produced pharmaceuticals. In this study, soluble expression of TNF-alpha in cytoplasm of E. coli was attempted using DsbC chaperon and the effects of environmental condition including inducer concentration and incubation temperature was evaluated.
METHODS: The gene encoding TNF-alpha was isolated by PCR reaction from cDNA that generated from PBMC total RNA and cloned into the pET-22b expression vector. Two type of E. coli strains, IPTG concentrations and growth temperatures as well as different type of media were examined with respect to the amount of recombinant protein produced in soluble form. Cytotoxicity assay on L929 cell line was performed for bioactivity evaluation.
RESULTS: Maximum amount of soluble TNF-alpha was attained by induction with 0.2 mM IPTG at 25 degrees C in the LB media. MTT assay with recombinant protein on L929 cells showed TNF-alpha expressed in active form with EC50 of 112 pg/mL.
CONCLUSIONS: This study report DsbC overexpressing strain (Shuffle (R) strain) markedly increases soluble expression of TNF-alpha in optimized condition.
- 出版日期2018-3
