Oxidative stress-resistance assay for screening yeast strains overproducing heterologous proteins

Many natural proteins have been developed into drugs and produced for direct application. Identifying improved hosts to achieve high-level heterologous protein production is a challenge in the study of heterologous protein expression in recombinant yeast. In this study, a novel high-throughput assay to screen such overproducing Saccharomyces cerevisiae strains was systematically developed. The protocol designed was based on screening host strain derivatives with increased superoxide dismutase dependent resistance to oxidative stress. Yeast cells transformed with recombinant plasmid carrying SOD1 gene as a reporter responded exquisitely to oxidative stress induced by elevated concentrations of paraquat. Improved yeast strains resulting from screening clones subjected to genome shuffling through selective pressure argue for a more effective screening system compared with traditonal selection. Moreover, this approach can be employed in general biochemical analysis without utilization of flow cytometry or well plate reader. Therefore, it is expected that the high-throughput assay would make superior strains producing heterologous proteins.     

A simple process for the isolation of epithelial cells from bacteria-contaminated samples using anchoring molecules

A simple and efficient tool to isolate epithelial cells from bacteria-contaminated samples has been developed using two different microparticles functionalized with chemical molecules. The epithelial cells could be captured simply by biocompatible anchors for membranes (BAM), consisting of poly(ethylene glycol) functionalized with oleyl-chain-conjugated NHS (N-hydroxysuccinimide) on glass microparticles, whereas bacteria were adsorbed on 3-aminopropyltrimethoxysilane (ATPS)-functionalized magnetic microparticles. In the case of samples highly contaminated with bacteria, epithelial cells were not isolated successfully by both of the single BAM- and antibody-functionalized microparticles. Therefore, serial isolation steps of these two different chemical functionalized microparticles were introduced. The concentration of bacteria was decreased dramatically by using APTS-functionalized magnetic particles prior to the isolation of epithelial cells by BAM microparticles. With these serial processes, successful isolation of epithelial cells was achieved from bacteria-contaminated epithelial samples. The applicability of this method was verified with bacteria-contaminated intestinal samples biopsied from a BALB/C mouse for primary cell cultivation.     

A Comprehensive Analysis of Symmetric Arginine Dimethylation in Colorectal Cancer Tissues Using Immunoaffinity Enrichment and Mass Spectrometry

Protein arginine methyltransferase 5 (PRMT5) is a major enzyme responsible for generating monomethyl and symmetric dimethyl arginine in proteins. PRMT5 is essential for cell viability and development, and its overexpression is observed in a variety of cancers. In the present study, it is found that levels of PRMT5 protein and symmetric arginine dimethylation in colorectal cancer (CRC) tissues are increased compared to those in adjacent noncancerous tissues. Using immunoaffinity enrichment of methylated peptides combined with high‐resolution mass spectrometry, a total of 147 symmetric dimethyl‐arginine (SDMA) sites in 94 proteins are identified, many of which are RNA binding proteins and enzymes. Quantitative analysis comparing CRC and normal tissues reveals significant increase in the symmetric dimethylation of 70 arginine sites in 46 proteins and a decrease in that of four arginine sites in four proteins. Among the 94 proteins identified in this study, it is confirmed that KH‐type splicing regulatory protein is a target of PRMT5 and highly expressed in CRC tissues compared to noncancerous tissues. This study is the first comprehensive analysis of symmetric arginine dimethylation using clinical samples and extends the number of known in vivo SDMA sites. The data obtained are available via ProteomeXchange with the identifier PXD015653.     

PDMS Microwell Stencil Based Multiplexed Single‐Cell Secretion Analysis

Multiplexed single‐cell protein secretion analysis provides an in‐depth understanding of cellular heterogeneity in intercellular communications mediated by secreted proteins in both fundamental and clinical research. However, it has been challenging to increase the proteomic parameters co‐profiled from every single cell in a facile way. Herein, a simple method to improve the multiplexed proteomic parameters of PDMS microwell based single‐cell secretion analysis platform by sandwiching PDMS stencil in between two antibody‐coated glass slides is introduced. Two different antibody panels can be immobilized easily by static coating, without using sophisticated fluid handling or bulky equipment. 5‐plexed, 3‐fluorescence color single‐cell secretion assay is demonstrated with this platform to investigate human monocytic U937 cells in response to lipopolysaccharide and phorbol myristate acetate stimulation, which identified the existence of functional subsets dictated by different cytokine profiles. The technology introduced here is simple, easy to operate, which holds great potential to become a powerful tool for profiling multiplexed single‐cell cytokine secretion at high throughput to dissect cellular heterogeneity in secretome signatures.