Engineering Saccharomyces Cerevisiae for the Production and Secretion of Affibody Molecules.

BackgroundAffibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to address if improvements in terms of yield, ease of production and if purification advantages can be identified. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. Results We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, showed to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind human HER3 as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. ConclusionThis study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching high yields and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.

Luteolin: A Dietary Molecule as Potential Anti-COVID-19 Agent

Luteolin (Lut) is an important plant-derived flavonoid that is widely distributed in edible herbs and vegetables. Studies on animal and human models have shown that Lut exhibits various pharmacological properties, viz. anti-inflammatory, anti-cancer, anti-oxidant, anti-apoptotic, and neurotrophic actions. The ongoing pandemic coronavirus disease-2019 (COVID-19), is a disease of the respiratory tract that consists of mild to severe symptoms of pneumonia including fever, muscle aches, sore throat, coughing, and shortness of breath. It is of particular concern in older people and patients with chronic diseases having cardiovascular and blood clotting issues or who have compromised immune. This situation prompted us to evaluate the bioactive compounds which are being used to prevent respiratory-related illness. Lut is one such compound which is used as an anti-inflammatory agent. Several studies have explained the protective nature of Lut by inhibiting virus entry and fusion with human receptors in old SARS-CoV that had emerged in 2003. Thus, regular consumption of foods having adequate amount of Lut in our diet may be helpful in inhibiting the SARS-CoV-2 infection as well and may prevent the consequent symptoms in COVID-19 patients. In present work, we have carried out the molecular docking studies of Lut with six different SARS-CoV-2 encoded key proteins. The FDA-approved drug remdesivir was also evaluated as control to compare the results. Lut showed excellent inhibitory action against papain-like proteinase, a main protease of SARS-CoV-2. Lut was also many times more active than remdesivir. Therefore, the foods which have Lut in adequate amount might be explored further for potential use against COVID-19

Identification of Down-Regulated Proteome in Saccharomyces cerevisiae with the Deletion of Yeast Cathepsin D in Response to Nitrogen Stress

Vacuolar proteinase A (Pep4p) is required for the post-translational precursor maturation of vacuolar proteinases in Saccharomyces cerevisiae, and important for protein turnover after oxidative damage. The presence of proteinase A in brewing yeast leads to the decline of beer foam stability, thus the deletion or inhibition of Pep4p is generally used. However, the influence of Pep4p deletion on cell metabolism in Saccharomyces cerevisiae is still unclear. Herein, we report the identification of differentially down-regulated metabolic proteins in the absence of Pep4p by a comparative proteomics approach. 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) presented that the number of significantly up-regulated spots (the Pep4p-deficient species versus the wild type) was 183, whereas the down-regulated spots numbered 111. Among them, 35 identified proteins were differentially down-regulated more than 10-fold in the Pep4p-deficient compared to the wild-type species. The data revealed that Pep4p was required for the synthesis and maturation of several glycolytic enzymes and stress proteins, including Eno2p, Fba1p, Pdc1p, Tpi1p, Ssa1, Hsp82p, and Trr1p. The transcription and post-translational modifications of glycolytic enzymes like Eno2p and Fba1p were sensitive to the absence of Pep4p; whereas the depletion of the pep4 gene had a negative impact on mitochondrial and other physiological functions. The finding of this study provides a systematic understanding that Pep4p may serve as a regulating factor for cell physiology and metabolic processes in S. cerevisiae under a nitrogen stress environment.