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Stress-induced degradation of the Bacillus subtilis anti-sigma factor RsiW results in the induction of genes controlled by the extracytoplasmic function sigma factor σW. RsiW is cleaved by the mechanism of regulated intramembrane proteolysis at site-1 and -2 by PrsW and RasP respectively, and is then further degraded by cytoplasmic Clp peptidases. In a reconstituted Escherichia coli system, PrsW removes 40 amino acids from RsiW by cleaving between Ala168 and Ser169 of the extracytoplasmic domain, thereby generating RsiW-S1. Further trimming of RsiW-S1's C-terminus by the periplasmic tail-specific protease Tsp is crucial for subsequent RasP-catalysed clipping. In B. subtilis, mutation of RsiW at Ala168 severely impairs site-1 processing. RsiW-S1 is undetectable in wild-type B. subtilis and knockout strains lacking various extracytoplasmic proteases. While it can be stabilized by C-terminal tagging, even this fusion protein is still attacked. Thus, several peptidases seem to be involved in trimming of RsiW downstream of PrsW and upstream of RasP in B. subtilis. Overall, the RsiW degradation pathway can be subdivided into two modules each consisting of a site-specific peptidase that prepares RsiW for further degradation by downstream proteases. [ABSTRACT FROM AUTHOR]

Human-derived CAP-T cell line has been demonstrated to be a powerful platform for high-titer production of HIV virus-like particles (VLPs) by PEI-mediated transient transfection. Scale-up of transfection processes is key to ensure the necessary quantities for pre-clinical and clinical testing. One of the major operational challenges of large-scale transient transfection is the medium replacement step that is often required before transfection. In this work, CAP-T cells were cultured in 1 L bioreactor with addition of sodium bicarbonate and surface aeration, which were observed to improve cell state for transfection. Remarkably, the medium replacement step was avoided by culturing the cells in a combination of media (FreeStyleF17 + 1% of PEM) compatible with cell growth and PEI-mediated transient transfection. In the conditions developed in this work, 0.5 × 10 6 cells/mL were seeded in 1 L bioreactor. Two days later, ∼2 × 10 6 cells/mL were transfected without medium exchange, using 0.5 pg of DNA/cell and 3 pg of PEI/cell. Transfection efficiency and VLP production comparable to shake flasks were obtained with a production of 4 × 10 10 VLPs/mL. This novel strategy significantly simplifies large-scale transient transfection, while suitable cell growth, transfection efficiency, and high quality VLP production are achieved. [ABSTRACT FROM AUTHOR]