Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of Escherichia coli DH5α-pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 °C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.

The widespread expression of therapeutic targets in both diseased and healthy tissues poses a major challenge for protein-based therapeutics, often leading to dose-limiting side effects. One promising strategy to enhance selectivity is reversible inactivation via affinity masks tethered through cleavable linkers responsive to disease-specific cues. Here, we introduce a workflow for the de novo design of peptide masks that reversibly inactivate miniprotein binders. By extending the C-terminus of the binder with a protease-cleavable linker and a masking helix, we generated minimal constructs that sterically block the receptor-binding interface. We applied this strategy to four therapeutically relevant targets, EGFR domains I and III, FGFR2, and IL7Rα, demonstrating broad applicability. Nearly half of the 20 designs achieved >100-fold affinity reduction, with the most effective mask decreasing EGFR binding by over 3 orders of magnitude. Upon cleavage by tumor-associated proteases, binding was restored in 19 out of 20 cases, confirming reversibility. We further show that micromolar or weaker affinity between the binder and the isolated mask is sufficient for robust inactivation and rapid activation. Additionally, by chemically conjugating a photocleavable linker, we created a light-responsive version of the masked binder, enabling external control with comparable efficiency to protease-sensitive designs. This work establishes a generalizable, rapid, and efficient platform for designing cleavable peptide masks from scratch, paving the way for conditionally active protein therapeutics responsive to endogenous or exogenous stimuli.

The widespread expression of many therapeutic targets in both diseased and healthy tissues presents a significant challenge for protein therapeutics, often resulting in dose-limiting side effects. To focus the action of biotherapeutics at the disease site, they can be reversibly inactivated by tethering an affinity mask through a linker cleavable by a disease-specific cue. However, current methods to generate affinity masks require extensive screening of masking sequence libraries. Here, we report a workflow to design the first de novo peptide masks for the reversible inactivation of miniprotein binders and we show its application on an EGFR antagonist miniprotein. By extending the C-terminus of the miniprotein to cover the binding interface, a masking sequence is generated that decreases binding to EGFR over 1000 times. Binding of the parental miniprotein can be fully rescued by cleavage of the linker between the binder and the mask with tumor-specific proteases. Additionally, by site-specifically conjugating the peptide mask through a chemical linker, we show we can render the protein responsive to alternative stimuli such as light. Our approach opens the doors to dramatically accelerating the development of affinity-based masks for other proteins with therapeutic potential to render them conditionally active to any stimulus.

The Lindau Nobel Laureate Meeting 2025 (LINO25) brought together more than 30 laureates and 600 young scientists in the field of chemistry from over 100 countries. One of the most remarkable contributions came from Latin America, with 24 young researchers from seven countries showcasing the high level, commitment, and diversity of science in the region. This special article documents the experience and contributions of this delegation, highlights their active role in scientific debates, their attainment of Nobel Alumni status, and their key recommendations for future generations. In addition, it provides a practical guide on the nomination process, logistical preparation, and strategies to maximize the experience during the event. Finally, it underlines the importance of increasing Latin America's visibility and strengthening its representation, including countries that have historically been underrepresented, in high-impact international scientific spaces such as Lindau.