RESUMO
RANKL/RANK/OPG axis is important in bone metabolism regulation, and becomes a popular research area in bone diseases. RANKL is a critical part of RANKL/RANK/OPG axis, and widely required in bone metabolism research. However, the yield of recombinant soluble human RANKL (hRANKL) in Escherichia coli is much lower than mouse RANKL (mRANKL). In this study, by adjusting and stabilizing the pH value of LB medium at 7.5, lowering the inducing temperature to 16 ℃ and optimizing the lysis program, the yield of soluble hRANKL increased by approximately 5 to 12-fold over the non-adjusted group. Our experiment effectively enhanced soluble hRANKL expression in E. coli and might constitute a meaningful attempt to obtain soluble expression of recombinant protein in E. coli.
RESUMO
To quantify the transcriptional activity of NF-κB and to screen drugs related to the regulation of NF-κB activation, we constructed a recombinant plasmid through deleting the original CMV promoter of retrovirus vector pQCXIP and inserting the NF-κB enhancer and NanoLuc luciferase sequence into the vector. Then, using the recombinant plasmid we constructed a cell line in which the expression of NanoLuc luciferase (NLuc) was regulated by NF-κB. The inserted sequences were verified by restriction endonuclease digestion and sequencing. Tumor necrosis factor-α (TNF-α), an NF-κB activator, acted on the constructed NLuc cell line and leaded to the specific luciferase reaction. The luciferase reaction showed a fine time and dose dependence to the TNF-α stimulation, indicating the successful construction of the NF-κB regulated NLuc-expressing cell line. Besides, the NF-κB inhibitor, triptolide, reduced the expression of NLuc in a dose-dependent way. The constructed reporter system in this study could be applied in the quantification of the NF-κB transcriptional activity and in the NF-κB regulation-related drug screening.