Project description:Background: Recombinant proteins are an important building block towards understanding the virulence mechanisms and pathophysiology of the major human pathogen Staphylococcus aureus. Of particular interest are secreted virulence factors. However, due to their potential toxicity and specific post-translational processing, virulence factors are difficult targets for heterologous protein production. Purified proteins with native conformation and satisfying purity can therefore often only be achieved by elaborate multi-step purification workflows. Homologous expression in S. aureus theoretically offers a beneficial alternative in this regard. However, due to the lack of systems with both tightly controlled expression and subsequent efficient purification, it is rarely used. Results: To bridge this gap, we present pTripleTREP as a versatile expression vector for S. aureus, which enables the homologous expression and purification of staphylococcal virulence factors. Based on a strong SigA-dependent staphylococcal promoter overlapped by three tetracycline responsive elements (TRE), the target gene promoter ensures tight repression under control conditions but high expression levels after induction. This allowed very precisely controlled production of the exemplary targets, serine protease-like protein A (SplA) and B (SplB). A simple single-step protein purification workflow via Twin-Strep-tag and Strep-Tactin®XT coated magnetic beads resulted in endotoxin-free Spl samples with purities above 99 %. The homologous production host, thereby, facilitates native secretion and maturation without the need to engineer the target gene sequence. Correct cleavage of the signal peptide and the corresponding enzymatic activity of the generated protein products were demonstrated with SplA and B. Conclusion: The expression vector pTripleTREP adds an important element to the staphylococcal molecular toolbox, facilitating the tightly controlled homologous expression and native but rapid purification of secreted staphylococcal virulence factors. The optimised architecture and genetic features of the vector additionally provide a good basis for further applications such as plasmid-based complementation or interaction studies. Thus, pTripleTREP will support research on the role of staphylococcal virulence factors aiming at future therapies to combat this pathogen.
Project description:Transcriptional profiling of mouse induced Treg (iTreg) cells comparing control (MIEG3) vector-transduced iTreg cells with iTreg cells transduced with YY1-overexpression vector. Tranduced cells were sorted by GFP expression. Goal was to determine the effects of YY1 on global iTreg gene expression.
Project description:In order to investigated the underlying molecular mechanism of GR’s effects, we transfected the Min6 cell with the GR cDNA expression vector or control vector, and then extracted RNA of Min6 cells for RNA sequencing. We studied the gene-expression differences resulting in GR overexpress. A total of 21542 gene transcripts was captured,and 233 genes showed significant expression differences (p-value<0.05,|log2Foldchange|>1) between the two group. Further GO analysis and KEGG analysis found that GR overactivation instigates dysregulated autophagy in beta-cells
