HT-29 and Caco-2 reporter cell lines for functional studies of nuclear factor kappa B activation.

The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1ß, and LPS) were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.

Expression of proteins in Pichia pastoris.

The methylotrophic yeast Pichia pastoris is currently one of the most versatile and popular hosts for the production of heterologous proteins, including industrial enzymes. The popularity of P. pastoris stems from its ability to grow to high cell densities, producing high titers of secreted heterologous protein with very low amounts of endogenous proteins. Its ability to express correctly folded proteins with post-translational modifications makes it an excellent candidate for the production of biopharmaceuticals. In addition, production in P. pastoris typically uses the strong, methanol-inducible and tightly regulated promoter (PAOX1), which can result in heterologous protein that constitutes up to 30% of total cell protein upon growth in methanol. In this chapter, we present methodology for the production of secreted recombinant proteins in P. pastoris, and we discuss alternatives to enhance protein production with the desired yield and quality.