Osmolyte-Mediated Encapsulation of Proteins inside MS2 Viral Capsids
| Autor: | Stacy L. Capehart, Danielle Tullman-Ercek, Matthew B. Francis, Jeff E. Glasgow |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Macromolecular Substances
Surface Properties viruses Molecular Conformation General Physics and Astronomy Heterologous Peptide Article Nanocapsules chemistry.chemical_compound Capsid Materials Testing Bacteriophage MS2 General Materials Science Levivirus chemistry.chemical_classification biology Chemistry Osmolar Concentration General Engineering Proteins biology.organism_classification Molecular biology In vitro Osmolyte Biophysics DNA |
| Zdroj: | ACS Nano. 6:8658-8664 |
| ISSN: | 1936-086X 1936-0851 |
| DOI: | 10.1021/nn302183h |
| Popis: | The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there are little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein stabilizing osmolyte, trimethylamine-N-oxide (TMAO), significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same Km value and a slightly lower kcat value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments. |
| Databáze: | OpenAIRE |
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