|Lin, Rui - NIH-NIEHS, NC|
|Ghosh, Sanjukta - NIH-NIEHS, NC|
Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 14, 2008
Publication Date: April 1, 2008
Citation: Cao, H., Lin, R., Ghosh, S., Anderson, R.A., Urban Jr, J.F. 2008. Production and characterization of ZFP36L1 antiserum against recombinant protein from Escherichia coli. Biotechnology Progress. 24(2)326-333. Interpretive Summary: Diet has been shown to play an important role in the prevention of inflammation-related diseases, such as arthritis, obesity, and type 2 diabetes. The diets commonly consumed in the United States and other developed countries appear to increase the incidence of these diseases. For the majority of the world population, drug treatment for these diseases is not feasible and alternative treatments need to be evaluated. Previous studies suggest that cinnamon and green tea extracts induce gene expression of tristetraprolin (TTP), a regulatory protein with anti-inflammatory function. TTP family proteins consist of three known members in mammals (TTP, ZFP36L1, and ZFP36L2) and the fourth member is in the mouse and rat X chromosome but not in humans (ZFP36L3). TTP is the best-studied family member. However, ZFP36L1 protein has not been adequately characterized, partly due to lack of high-titer antibodies and purified protein. To understand the molecular and biochemical properties of this protein, mouse ZFP36L1 was overexpressed in E. coli, the recombinant protein purified, and then used for the production of polyclonal antibodies. ZFP36L1 antiserum and the anti-MBP-mTTP antiserum obtained from rabbits were used to detect these two proteins in mouse macrophages and adipocytes following treatments with insulin, lipopolysaccharide, and cinnamon polyphenols. Real-time PCR was used to estimate the relative abundance of both mRNA levels in these cells. The results indicate that TTP and ZFP36L1 are differentially expressed at the mRNA and protein levels in these mouse cells. These findings should be of interest to scientists.
Technical Abstract: Tristetraprolin (TTP/ZFP36) family proteins are anti-inflammatory. They bind and destabilize some AU-rich element-containing mRNAs such as tumor necrosis factor mRNA. In this study, recombinant ZFP36L1/TIS11B (a TTP homologue) was over-expressed in E. coli, purified, and used for polyclonal antibody production in rabbits. This antiserum and the antiserum against recombinant mouse TTP were used to detect ZFP36L1 and TTP in mouse 3T3-L1 adipocytes and RAW264.7 cells. Immunoblotting showed that ZFP36L1 was stably expressed, but TTP was induced by insulin and cinnamon, and not by lipopolysaccharide (LPS) in 3T3-L1 adipocytes. In contrast, ZFP36L1 was undetectable, but TTP was strongly induced in LPS-stimulated RAW264.7 cells. Quantitative real-time polymerase chain reaction (PCR) confirmed the higher levels of ZFP36L1 mRNA in adipocytes and TTP mRNA in RAW264.7 cells. Low levels of ZFP36L1 expression were also confirmed by northern blotting in mouse embryonic fibroblasts. These results demonstrate that ZFP36L1 antiserum is useful in the detection of this protein, and that TTP and ZFP36L1 are differentially expressed at the mRNA and protein levels in mouse adipocytes and macrophages.