Effect of the Protein Chain Conformation on the Collapse into Nanoparticles.

Protein single-chain nanoparticles can outperform synthetic nanoparticles in biomedical applications due to enhanced biocompatibility. Compared to synthetic (co)polymers, the chemical complexity of proteins challenges chain conformation control. Here, we investigate the impact of the precursor chain conformation of bovine serum albumin (BSA) on the nanoparticle structure after intramolecular cross-linking. We explore the urea concentration (denaturant), pH, salt, cross-linker length, and concentration. Small-angle neutron scattering and dynamic light scattering experiments reveal a shrinking chain conformation upon cross-linking. However, the ability to collapse depends on solvent conditions: more expanded chains collapse more, whereas proteins that are already compact barely change in size upon cross-linking. Static light scattering measurements demonstrate that binding is primarily intramolecular. The use of a shorter cross-linker does not lead to collapse of extended chains. Overall, BSA exhibits a similar behavior to that of polymer nanoparticles, which then allows to harness the precursor conformation for morphological control.

Investigation of variants in genetics and virulence of Porcine Epidemic Diarrhea Virus after serial passage on Vero cells.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious intestinal virus. However, the current PEDV vaccine, which is produced from classical strain G1, offers low protection against recently emerged strain G2. This study aims to develop a better vaccine strain by propagating the PS6 strain, a G2b subgroup originating from Vietnam, on Vero cells until the 100th passage. As the virus was propagated, its titer increased, and its harvest time decreased. Analysis of the nucleotide and amino acid variation of the PS6 strain showed that the P100PS6 had 11, 4, and 2 amino acid variations in the 0 domain, B domain, and ORF3 protein, respectively, compared to the P7PS6 strain. Notably, the ORF3 gene was truncated due to a 16-nucleotide deletion mutation, resulting in a stop codon. The PS6 strain's virulence was evaluated in 5-day-old piglets, with P7PS6 and P100PS6 chosen for comparison. The results showed that P100PS6-inoculated piglets exhibited mild clinical symptoms and histopathological lesions, with a 100% survival rate. In contrast, P7PS6-inoculated piglets showed rapid and typical clinical symptoms of PEDV infection, and the survival rate was 0%. Additionally, the antibodies (IgG and IgA) produced from inoculated piglets with P100PS6 bound to both the P7PS6 and P100PS6 antigens. This finding suggested that the P100PS6 strain was attenuated and could be used to develop a live-attenuated vaccine against highly pathogenic and prevalent G2b-PEDV strains.

Neat Protein Single-Chain Nanoparticles from Partially Denatured BSA.

The main challenge for the preparation of protein single-chain nanoparticles (SCNPs) is the natural complexity of these macromolecules. Herein, we report the suitable conditions to produce "neat" bovine serum albumin (BSA) single-chain nanoparticles (SCNPs) from partially denatured BSA, which involves denaturation in urea and intramolecular cross-linking below the overlap concentration. We use two disuccinimide ester linkers containing three and six methylene spacer groups: disuccinimidyl glutarate (DSG) and disuccinimidyl suberate (DSS), respectively. Remarkably, the degree of internal cross-linking can be followed simply and efficiently via 1H NMR spectroscopy. The associated structural changes-as probed by small-angle neutron scattering (SANS)-reveal that the denatured protein has a random-like coil conformation, which progressively shrinks with the addition of DSG or DSS, thus allowing for size control of the BSA-SCNPs with radii of gyration down to 5.4 nm. The longer cross-linker exhibits slightly more efficiency in chain compaction with a somewhat stronger size reduction but similar reactivity at a given cross-linker concentration. This reliable method is applicable to a wide range of compact proteins since most proteins have appropriate reactive amino acids and denature in urea. Critically, this work paves the way to the synthesis of "neat", biodegradable protein SCNPs for a range of applications including nanomedicine.