Abstract
In cellular systems, compartmentalization plays an important role in the protection and regulation of enzymes. Controlled encapsulation of enzymes in nanocompartments is crucial in understanding biocatalytic processes in the cellular environment. We have recently described an enzymatic method to covalently attach enzymes, equipped with a small recognition peptide, to the interior of viral capsids. Viral capsids are especially interesting in this respect, as they form very well-defined nanoparticles with a uniform size and shape. Here, we describe the relevant experimental procedures to encapsulate a model enzyme into the interior of a viral capsid, purify the resulting viral capsids, and measure the catalytic activity of the encapsulated enzymes.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Schoonen L, van Hest JCM (2014) Functionalization of protein-based nanocages for drug delivery applications. Nanoscale 6:7124–7141
Caspar DLD, Klug A (1962) Physical principles in the construction of regular viruses. Cold Spring Harb Symp Quant Biol 27:1–24
Speir JA, Munshi S, Wang G et al (1995) Structures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscopy. Structure 3:63–78
Schoonen L, Pille J, Borrmann A et al (2015) Sortase A-mediated N-terminal modification of cowpea chlorotic mottle virus for highly efficient cargo loading. Bioconjug Chem 26:2429–2434
Clancy KW, Melvin JA, McCafferty DG (2010) Sortase transpeptidases: insights into mechanism, substrate specificity, and inhibition. Biopolymers 94:385–396
Schoonen L, Nolte RJM, van Hest JCM (2016) Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic. Nanoscale 8:14467–14472
van Eldijk MB, Wang JC-Y, Minten IJ et al (2012) Designing two self-assembly mechanisms into one viral capsid protein. J Am Chem Soc 134:18506–18509
Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326
Ton-That H, Liu G, Mazmanian SK et al (1999) Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci 96:12424–12429
Acknowledgments
The Ministry of Education, Culture, and Science (Gravitation program 024.001.035) is acknowledged for financial support.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Schoonen, L., van Hest, J.C.M. (2018). Modification of CCMV Nanocages for Enzyme Encapsulation. In: Udit, A. (eds) Protein Scaffolds. Methods in Molecular Biology, vol 1798. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7893-9_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7893-9_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7892-2
Online ISBN: 978-1-4939-7893-9
eBook Packages: Springer Protocols