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Development of a Hepatitis B virus capsid as a nanocontainer and a carrier for protein delivery into cells

Dahlin, Jonathan LU (2016) KEMT30 20132
Department of Chemistry
Abstract
Encapsulation systems have long been studied, and the designs have varied in both capsule material as well as the type of cargo. The intended use have ranged from nanoreactors to delivery systems.
This work presents an encapsulation system consisting of protein capsule and a protein cargo. The capsule is a redesigned hepatitis B virus capsid protein, in which the luminal extension has been altered to bind the protein calmodulin instead of the viral genome. The cargo is consequently a calmodulin fused protein.
It is shown that co-expression of the redesigned capsid protein together with a capsid protein lacking a luminal extension greatly aids the solubility and assembly of redesigned capsids. Furthermore it is shown that the approximate... (More)
Encapsulation systems have long been studied, and the designs have varied in both capsule material as well as the type of cargo. The intended use have ranged from nanoreactors to delivery systems.
This work presents an encapsulation system consisting of protein capsule and a protein cargo. The capsule is a redesigned hepatitis B virus capsid protein, in which the luminal extension has been altered to bind the protein calmodulin instead of the viral genome. The cargo is consequently a calmodulin fused protein.
It is shown that co-expression of the redesigned capsid protein together with a capsid protein lacking a luminal extension greatly aids the solubility and assembly of redesigned capsids. Furthermore it is shown that the approximate 1:1 ratio resulting from the co-expression is maintained throughout disassembly and reassembly of the capsids. (Less)
Popular Abstract
Scientists have long been interested in so called encapsulation systems, i.e. the ability to enclose a certain molecule inside a shell or capsule composed of another type molecule. Many different types of capsules have been suggested, from lipids to DNA to proteins. The enclosed molecule, often referred to as the cargo, has also been quite varied, ranging from small organic molecules to nucleic acids and proteins.
There's generally two main applications for these encapsulation systems, nanoreactors and delivery systems. For nanoreactors the idea would be to encapsulate one or several enzymes within the capsule and then study enzymatic properties or leverage the nature of the capsule achieve a greater environmental control. The idea for... (More)
Scientists have long been interested in so called encapsulation systems, i.e. the ability to enclose a certain molecule inside a shell or capsule composed of another type molecule. Many different types of capsules have been suggested, from lipids to DNA to proteins. The enclosed molecule, often referred to as the cargo, has also been quite varied, ranging from small organic molecules to nucleic acids and proteins.
There's generally two main applications for these encapsulation systems, nanoreactors and delivery systems. For nanoreactors the idea would be to encapsulate one or several enzymes within the capsule and then study enzymatic properties or leverage the nature of the capsule achieve a greater environmental control. The idea for the delivery systems would be to encapsulate a molecule of interest such as a drug or piece of DNA, and use the capsule as a vehicle for delivery into living cells. The capsule could serve a dual purpose in both protecting the loaded cargo from the external environment as well as providing a targeting and entry mechanism into the cells.

In this work an encapsulation system based on a protein capsule and a protein cargo is used. More specifically the protein capsule is based of the capsid protein from the Hepatitis B virus (HBV). In nature the HBV capsid protein bind to and form a capsid structure around the viral genome. The genome binding is carried out through binding interaction with a peptide extension extending into the interior or lumen of the capsid from the main capsid protein body. In this system the capsid protein have been redesigned by replacing this luminal genome binding peptide with a protein binding peptide. By fusing the intended cargo with a peptide binding protein it is possible to achieve specific encapsulation of the desired cargo. Furthermore, the peptide-protein pair have been chosen in such a way that their binding interaction can be modulated by the presence or absence of calcium ions, allowing the cargo to dissociate from the capsid wall after encapsulation.

One of the key functions in an encapsulation system is the ability to form the surrounding shell (here capsid). However, even small changes in proteins can have great effects on their characteristics. It had previously been shown that removing the luminal genome binding peptide of the HBV capsid proteins still allowed the formation of functional capsids. But it was shown here that simply replacing the peptide with a protein binding peptide resulted in non-functional capsid formation.
However, by simultaneously expressing both the redesigned capsid protein with the capsid protein lacking the luminal peptide functional capsids could be obtained. These functional capsids was composed of a roughly 1:1 mixture of the two protein variants, furthermore this mixed composition was maintained throughout disassembly and reassembly of the capsids. The current hypothesis is that the presence of proteins without a luminal peptide reduces clashing between the peptide extensions in the tight spaces of the capsid lumen.
This concept of co-expression and clashing reduction is probably the most important take away message from this work, and could potentially be applied to some other systems where clashing between subunits is a problem. (Less)
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author
Dahlin, Jonathan LU
supervisor
organization
course
KEMT30 20132
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Hepatitis B Virus, Protein Design, protein engineering, HBV, protein science, proteinvetenskap
language
English
id
8871028
date added to LUP
2016-04-21 10:33:50
date last changed
2016-04-21 10:33:50
@misc{8871028,
  abstract     = {Encapsulation systems have long been studied, and the designs have varied in both capsule material as well as the type of cargo. The intended use have ranged from nanoreactors to delivery systems.
This work presents an encapsulation system consisting of protein capsule and a protein cargo. The capsule is a redesigned hepatitis B virus capsid protein, in which the luminal extension has been altered to bind the protein calmodulin instead of the viral genome. The cargo is consequently a calmodulin fused protein.
It is shown that co-expression of the redesigned capsid protein together with a capsid protein lacking a luminal extension greatly aids the solubility and assembly of redesigned capsids. Furthermore it is shown that the approximate 1:1 ratio resulting from the co-expression is maintained throughout disassembly and reassembly of the capsids.},
  author       = {Dahlin, Jonathan},
  keyword      = {Hepatitis B Virus,Protein Design,protein engineering,HBV,protein science,proteinvetenskap},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of a Hepatitis B virus capsid as a nanocontainer and a carrier for protein delivery into cells},
  year         = {2016},
}