Biology subjects

Jerala, R.

Publications and source records attributed to Jerala, R..

3 recordsLinked to original sources

bioRxiv2020

Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion

Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism and screen inhibition of viral spike protein-mediated fusion under the biosafety level 1conditions.

synthetic biology↗

bioRxiv2020

A Multiplexed Bacterial Two-Hybrid for Rapid Characterization of Protein-Protein Interactions and Iterative Protein Design

Myriad biological functions require protein-protein interactions (PPIs), and engineered PPIs are crucial for applications ranging from drug design to synthetic cell circuits. Understanding and engineering specificity in PPIs is particularly challenging as subtle sequence changes can drastically alter specificity. Coiled-coils are small protein domains that have long served as a simple model for studying the sequence-determinants of specificity and have been used as modular building blocks to build large protein nanostructures and synthetic circuits. Despite their simple rules and long-time use, building large sets of well-behaved orthogonal pairs that can be used together is still challenging because predictions are often inaccurate, and, as the library size increases, it becomes difficult to test predictions at scale. To address these problems, we first developed a method called the Next-Generation Bacterial Two-Hybrid (NGB2H), which combines gene synthesis, a bacterial two-hybrid assay, and a high-throughput next-generation sequencing readout, allowing rapid exploration of interactions of programmed protein libraries in a quantitative and scalable way. After validating the NGB2H system on previously characterized libraries, we designed, built, and tested large sets of orthogonal synthetic coiled-coils. In an iterative set of experiments, we assayed more than 8,000 PPIs, used the dataset to train a novel linear model-based coiled-coil scoring algorithm, and then characterized nearly 18,000 interactions to identify the largest set of orthogonal PPIs to date with twenty-two on-target interactions.

molecular biology↗

bioRxiv2020

Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and {beta}-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue {beta}-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.

immunology↗