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Coproduction of multiple proteins at high levels in a single human cell line would be extremely useful for basic research and medical applications. Here, a novel strategy for the stable expression of multiple proteins by integrating the genes into defined transcriptional hotspots in the human genome is presented. As a proof-of-concept, it is shown that EYFP is expressed at similar levels from hotspots and that the EYFP expression increases proportionally with the copy number. It is confirmed that three different fluorescent proteins, encoded by genes integrated at different loci, can be coexpressed at high levels. Further, a stable cell line is generated, producing antigens from different human coronaviruses: MERS-CoV and HCoV-OC43. Antibodies raised against these antigens, which contain human N-glycosylation, show neutralizing activities against both viruses, suggesting that the coexpression system provides a quick and predictable way to produce multiple coronavirus antigens, such as the recent 2019 novel human coronavirus. [ABSTRACT FROM AUTHOR]

One of important issues in protein science is to increase the protein stability. Utilizing a ketosteroid isomerase (KSI) as a model system, we constructed a mutant KSI which has a higher stability than its WT. We focused on the role of aromatic moieties of three tyrosine residues, Tyr14, Tyr55, and Tyr30, which constitute a hydrogen bond network in KSI. Therefore, we made a series of mutants which could substitute tyrosine with phenylalanine to remove the hydroxyl group in the tyrosine residue. We measured the free-energy change for unfolding using urea as a denaturant. We investigated the interaction among these aromatic residues for stability using the double mutant cycle analysis. We found that additional mutations on Y14F, Y55F, and Y14F/Y55F, respectively, by replacement of tyrosine at the position 30 with phenylalanine increased the stability ca. 4.0 kcal/mol. Especially the triple mutant KSI (Y14F/Y30F/Y55F) was more stable 2 kcal/mol than wild type KSI. Our structural analyses suggests that the enhanced stability of the triple mutant should be due to increased both hydrophobic interaction and aromatic-aromatic interaction upon the mutation [ABSTRACT FROM AUTHOR]