Expression and purification of the human epidermal growth factor receptor extracellular domain.

In the fields of drug discovery and protein science, small quantities of proteins are always needed to investigate or validate protein-protein (or protein-small molecule) interactions. Traditional transient or stable expression method to obtain recombinant proteins in eukaryotic systems can be laborious and time-consuming, especially when multiple protein variants are required. Here, we present a fast and convenient method for obtaining small quantities of recombinant human epidermal growth factor receptor (rhEGFR) ectodomain protein, which could be efficiently extended to the expression of other eukaryotic proteins. Human EGFR ectodomain gene was inserted into the plasmid pBMN-GFP and recombinant plasmid was transfected into HEK 293 T cells. In the presence of hygromycin, cells with the integrated human EGFR ectodomain gene were selected and proliferated. rhEGFR ectodomain in cell culture supernatant was purified using serial connected diethylaminoethyl Sepharose column and Ni-NTA Sepharose column. Purity of the final purified rhEGFR ectodomain was over 95% according to SDS-PAGE analysis. Moreover, the purified target protein was biological active via measuring the affinity between the rhEGFR ectodomain and rhEGF. Our method could greatly facilitate research in the areas of protein science, protein structural biology, and drug discovery.

Secretory expression of negative regulatory region of human Notch1 in Escherichia coli and preparation of a functional polyclonal antibody.

Notch signaling is a highly conserved pathway existed in multicellular organisms. It plays roles in normal human body development, human cancer initiation, progression and metastasis. The Notch negative regulatory region (NRR) is critical for Notch signaling, and cleavage at the S2 site in the NRR ultimately leads to the activation of Notch signaling. To study the function of human NRR1, we expressed the recombinant human NRR1 (rhNRR1) domain in Escherichia coli. After purification, rhNRR1 was obtained with approximately 94% purity according to SDS-PAGE analysis. Furthermore, the polyclonal anti-rhNRR1 serum raised by immunizing mouse with the purified rhNRR1 was able to reduce the generation of active form of Notch1 intracellular domain in HeLa cells, which implied the raised antibody could recognize and bind the natural conformation of Notch1 NRR. Preparation of rhNRR1 by this way is convenient, time-consuming, and could be used to the preparation of anti-NRR1 therapeutic antibody.

Triclosan inhibits testosterone biosynthesis in adult rats via inducing m6A methylation-mediated autophagy.

Triclosan is a potent antibacterial compound widely used in everyday products. Whether triclosan affects Leydig cell function in adult male rats remains unknown. In this study, 0, 50, 100, or 200 mg/kg/day triclosan was gavaged to Sprague-Dawley male rats from 56 to 63 days postpartum. Triclosan significantly reduced serum testosterone levels at ≥ 50 mg/kg/day via downregulating the expression of Leydig cell gene Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, and Hsd17b3 and regulatory transcription factor Nr3c2 at 100-200 mg/kg. Further analysis showed that triclosan markedly increased autophagy as shown by increasing LC3II and BECN1 and decreasing SQSTM1. The mRNA m6A modification analysis revealed that triclosan significantly downregulated Fto expression at 200 mg/kg while upregulating Ythdf1 expression at 100 and 200 mg/kg, leading to methylation of Becn1 mRNA as shown by MeRIP assay. Triclosan significantly inhibited testosterone output in rat R2C Leydig cells at ≥ 5 µM via downregulating Fto and upregulating Ythdf1. SiRNA Ythdf1 knockdown can reverse triclosan-mediated mitophagy in R2C cells, thereby reversing the reduction of testosterone output. In summary, triclosan caused Becn1 m6A methylation by downregulating Fto and upregulating Ythdf1, which accelerated Becn1 translation, thus leading to the occurrence of autophagy and the decrease of testosterone biosynthesis.

Synthesis and in vitro biological evaluation of novel dendrocandin analogue as potential anti-tumor agent.

Dendrocandins are characteristic chemical structures of D. officinale and have strong physiological bioactivities. In this study, a dendrocandin analogue (1) has been prepared by total synthesis (9 steps, 12.6% overall yield) in which coupling reaction and Wittig reaction as the key steps. Compound 1 was also evaluated for its anticancer activity in vitro against six human cancer cells (MCF-7, A549, A431, SW480, HepG-2 and HL-60) using MTT assays. Compound 1 showed potent cytotoxicity, with the IC50 value 16.27 ± 0.26 µM. The expression levels of apoptotic proteins indicated that compound 1 can up-regulate the expression of apoptotic proteins, leading to apoptosis. This compound suggested that it's potential as anticancer agent for further development.