Dihydroartemisinin enhances VEGFR1 expression through up-regulation of ETS-1 transcription factor.

Angiogenesis is required for tumor growth. Dihydroartemisinin (DHA), a the effective anti-malarial derivative of artemisinin, demonstrated potent anti-angiogenic activities that closely related to the regulation of vascular endothelial growth factor (VEGF) signaling cascade. VEGF receptor 1 (VEGFR1), a receptor in endothelial cells (ECs), coordinately regulate angiogenic activity triggered by ligand-receptor binding. Here we aimed to explore the effects of DHA on VEGFR1 expression in ECs. We found that DHA significantly increases VEGFR1 expression in human umbilical vein endothelial cells (HUVECs). In addition, DHA significantly upregulates the level of V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1 (ETS-1), a transcriptional factor which binds to the human VEGFR1 promoter. ChIP assay showed that DHA increases ETS-1 binding to the -52 ETS motif on the VEGFR1 promoter. Knockdown of ETS-1 by RNA interference abolished DHA-induced increase of VEGFR1 expression. Taken together, we demonstrated that DHA elevates VEGFR1 expression via up-regulation of ETS-1 transcription in HUVECs.

Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (ZBpa-Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with ZBpa-Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the ZBpa-Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL-1, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL-1). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications.

Site-specific, covalent immobilization of BirA by microbial transglutaminase: A reusable biocatalyst for in vitro biotinylation.

A facile approach for the production of a reusable immobilized recombinant Escherichia coli biotin ligase (BirA) onto amine-modified magnetic microspheres (MMS) via covalent cross-linking catalyzed using microbial transglutaminase (MTG) was proposed in this study. The site-specifically immobilized BirA exhibited approximately 95% of enzymatic activity of the free BirA, and without a significant loss in intrinsic activity after 10 rounds of recycling (P > 0.05). In addition, the immobilized BirA can be easily recovered from the solution via a simple magnetic separation. Thus, the immobilized BirA may be of general use for in vitro biotinylation in an efficient and economical manner.

An efficient protocol to enhance the extracellular production of recombinant protein from Escherichia coli by the synergistic effects of sucrose, glycine, and Triton X-100.

Targeting recombinant proteins at highly extracellular production in the culture medium of Escherichia coli presents a significant advantage over cytoplasmic or periplasmic expression. In this work, a recombinant protein between ZZ protein and alkaline phosphatase (rZZ-AP) was constructed. Because rZZ-AP has the IgG-binding capacity and enzymatic activity, it can serve as an immunoreagent in immunoassays. However, only a very small portion of rZZ-AP is generally secreted into the aqueous medium under conventional cultivation procedure. Hence, we emphasized on the optimization of the culture procedures and attempted to dramatically enhance the yield of extracellular rZZ-AP from E. coli HB101 host cells by adding sucrose, glycine, and Triton X-100 in the culture medium. Results showed that the extracellular production of rZZ-AP in the culture medium containing 5% sucrose, 1% glycine, and 1% Triton X-100 was 18.6 mg/l, which was 18.6-fold higher than that without the three chemicals. And the ß-galactosidase activity test showed that the increased extracellular rZZ-AP was not due to cell lysis. Further analysis suggested a significant interaction effect among the three chemicals for the enhancement of extracellular production. Ultrastructural analysis indicated that the enhancement may be due to the influence of sucrose, glycine, and Triton X-100 on the periplasmic osmolality, permeability, or integrity of the cell wall, respectively. This proposed approach presents a simple strategy to enhance the extracellular secretion of recombinant proteins in the E. coli system at the process of cell cultivation.