The packaging of active enzymes in protein cages is a powerful strategy to control catalytic activity. Using a positively supercharged variant of green fluorescent protein, GFP(+36), as a genetically programmable tag, enzymes can be rapidly and quantitatively loaded into an engineered variant of the Aquifex aeolicus cage-forming protein lumazine synthase (AaLS-13) that possesses a negatively charged lumen. The cargo is spontaneously localized within AaLS-13 cages by simply mixing the components in aqueous solution. This chapter describes a detailed protocol for the preparation of AaLS-13 cages and GFP(+36)-enzyme fusions, as well as characterization of the inclusion complexes. Suitable conditions for encapsulation and enzyme kinetic assays are also discussed.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-4939-7893-9_4 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Combination application of Pickering emulsion and BLS protein nanoparticles enhances vaccine efficacy.
Int J Biol Macromol
December 2024
Key Laboratory of Livestock Infectious Diseases in Northeast China, Ministry of Education, College of Animal Science and Veterinary Medicine, Shenyang Agricultural University. Liaoning Province, Shenyang 110866, PR China; NMPA Key Laboratory for Quality Monitoring and Evaluation of Vaccines and Biological Products, Key Laboratory of Tropical Diseases Control, School of public health, Sun Yat-sen University, Guangzhou 510275, PR China; Collaborative Innovation Center for Prevention and Control of Zoonoses, Jinzhou Medical University, Jinzhou 121001, PR China. Electronic address:
The deficiency of recombinant protein immune response could be compensated for by using nanoparticle platforms or adding immune enhancers, however existing vaccines or adjuvants struggle to elicit durable cellular immune responses. In this work, a protein nanoscaffold, lumazine synthase isolated from Brucella (BLS) was optimally designed that could facilitate cellular uptake of displayed antigens and the maturation of bone marrow-derived dendritic cells (BMDCs), and enhancing humoral immune responses. To enhance cellular immune response, chitosan hydrochloride-stabilized Pickering emulsion (CHSPE) was evaluated as an adjuvant for the BLS nanoscaffold-based vaccine.
View Article and Find Full Text PDFEvaluation of SARS-CoV-2-Specific IgY Antibodies: Production, Reactivity, and Neutralizing Capability against Virus Variants.
Int J Mol Sci
July 2024
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, 17493 Greifswald, Germany.
The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel.
View Article and Find Full Text PDFStructure of Aquifex aeolicus lumazine synthase by cryo-electron microscopy to 1.42 Å resolution.
IUCrJ
September 2024
Biological and Environmental Science and Engineering, King Abdullah University of Science and Technology, 4700 KAUST, Thuwal 23955, Saudi Arabia.
Single-particle cryo-electron microscopy (cryo-EM) has become an essential structural determination technique with recent hardware developments making it possible to reach atomic resolution, at which individual atoms, including hydrogen atoms, can be resolved. In this study, we used the enzyme involved in the penultimate step of riboflavin biosynthesis as a test specimen to benchmark a recently installed microscope and determine if other protein complexes could reach a resolution of 1.5 Å or better, which so far has only been achieved for the iron carrier ferritin.
View Article and Find Full Text PDFProtection against the H1N1 influenza virus using self-assembled nanoparticles formed by lumazine synthase and bearing the M2e peptide.
Virology
September 2024
National Key Laboratory of Agricultural Microbiology, Hubei Hongshan Laboratory, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, Hubei, China. Electronic address:
There is an urgent need for influenza vaccines that offer broad cross-protection. The highly conserved ectodomain of the influenza matrix protein 2 (M2e) is a promising candidate; however, its low immunogenicity can be addressed. In this study, we developed influenza vaccines using the Lumazine synthase (LS) platform.
View Article and Find Full Text PDFProtein Nanoparticles as Vaccine Platforms for Human and Zoonotic Viruses.
Viruses
June 2024
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA.
Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!