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Caffeic acid phenethyl ester (CAPE), a natural polyphenol derived from propolis, has a good regulatory effect on lipid metabolism, but its molecular mechanism is not clear. In the present study, the ameliorative effect of CAPE on lipid metabolism in oleic acid-induced HepG2 human liver cancer cells and its mechanism at the cellular level were investigated. The results showed that compared with the high fat group, lipid accumulation in the cells was significantly improved after CAPE intervention, and a total of 3 270 differentially expressed genes (DEGs) were selected at the transcriptomic level; 1 351 of them were up-regulated and the rest were down-regulated. The functional annotation analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database showed that the DEGs caused by CAPE intervention were significantly annotated to lipid metabolism-related pathways. The KEGG signaling pathway enrichment analysis showed that the HIF-1α pathway and the fatty acid catabolism pathway were significantly enriched. The expression levels of gene HIF-1α, PPARα, CPT1A and FABP5 were 1.326, 1.661, 2.039 and 2.598 times higher in the CAPE group than the high fat group. It is suggested that CAPE can improve lipid metabolism disorder in oleic acid-induced cells possibly by reducing oxidative stress and regulating the PPARα and fatty acid oxidative catabolic pathways. This study provides a theoretical reference for future research on the molecular mechanism of lipid metabolism regulation by CAPE and the regulation of lipid metabolism disorder induced by a high-fat diet.

Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.