Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system improved light-induced dimer (iLID), which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli , the blue light-sensitive part of iLID, As LOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to As LOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the POI, which could potentially facilitate other optogenetic manipulations of protein–protein interaction.
Radiation-induced hair cell injury is detrimental for human health but the underlying mechanism is not clear. MicroRNAs (miRNAs) have critical roles in various types of cellular biological processes. The present study investigated the role of miR-222 in the regulation of ionizing radiation (IR)-induced cell injury in auditory cells and its underlying mechanism. Real-time PCR was performed to identify the expression profile of miR-222 in the cochlea hair cell line HEI-OC1 after IR exposure. miRNA mimics or inhibitor-mediated up- or down-regulation of indicated miRNA was applied to characterize the biological effects of miR-222 using MTT, apoptosis and DNA damage assay. Bioinformatics analyses and luciferase reporter assays were applied to identify an miRNA target gene. Our study confirmed that IR treatment significantly suppressed miR-222 levels in a dose-dependent manner. Up-regulation of miR-222 enhances cell viability and alleviated IR-induced apoptosis and DNA damage in HEI-OC1 cells. In addition, BCL-2-like protein 11 (BCL2L11) was validated as a direct target of miR-222. Overexpression of BCL2L11 abolished the protective effects of miR-222 in IR-treated HEI-OC1 cells. Moreover, miR-222 alleviated IR-induced apoptosis and DNA damage by directly targeting BCL2L11. The present study demonstrates that miR-222 exhibits protective effects against irradiation-induced cell injury by directly targeting BCL2L11 in cochlear cells.
The alternation of light and dark rhythm causes a series of physiological, biochemical and metabolic changes in animals, which also alters the growth and development of animals, and feeding, migration, reproduction and other behavioral activities. In recent years, many studies have reported the effects of long-term (more than 6 weeks) illumination on ovarian growth and development. In the present study, we observed the damage, repair and apoptosis of ovarian DNA in a short period of illumination. The results showed that, in short time (less than 2 weeks) illumination conditions, the 24-h light treatment caused the reduction of total ovarian follicle number and down-regulation of circadian clock related genes. Furthermore, the changed levels of serum sex hormones were also detected after 24-h light exposure, of which the concentrations of LH (luteinizing hormone), FSH (follicle-stimulating hormone) and E2 (estradiol) were increased, but the concentration of PROG (progesterone) was decreased. Moreover, 24-h light exposure increased the expression of DNA damage and repair related genes, the number of TUNEL and RAD51 positive cells. These results indicated that 24-h light exposure for 4, 8 and 12 days increased DNA damage and cell apoptosis, thereby affecting the development of ovary.