Advanced

Purification, crystallization and characterization of modified HBV capsid

Tupiņa, Dagnija (2015) MOBN01 20151
Degree Projects in Molecular Biology
Abstract
Nanocarriers and nanoreactors are often derived from viral capsids. The capsid of HBV is a particularly attractive self-assembling model, fit for conversion into protein carrier. In this thesis modified HBV capsid is expressed in E.coli and purified by size exclusion chromatography. Nanocarrier system is based on principle of specific binding between the modified capsid building block and protein cargo. Binding of cargo and modified capsid building block is determined by fluorescence spectroscopy and binding constant is determined. Crystallization screen is performed to solve the structure and determine the position of the added tag. This will evaluate the possibility of seeing the structure of the cargo as well. In addition, self-assembly... (More)
Nanocarriers and nanoreactors are often derived from viral capsids. The capsid of HBV is a particularly attractive self-assembling model, fit for conversion into protein carrier. In this thesis modified HBV capsid is expressed in E.coli and purified by size exclusion chromatography. Nanocarrier system is based on principle of specific binding between the modified capsid building block and protein cargo. Binding of cargo and modified capsid building block is determined by fluorescence spectroscopy and binding constant is determined. Crystallization screen is performed to solve the structure and determine the position of the added tag. This will evaluate the possibility of seeing the structure of the cargo as well. In addition, self-assembly conditions are explored to potentially improve the encapsidation. (Less)
Popular Abstract
Molecular nano-carrier of proteins

Nature is using proteins to create an unfathomably rich variety of structures and functions. Some of them can be ‘’stolen’’ and adjust them for purposes and needs of science and humanity. One such function is a need for nanostructure that could contain enclose molecules and proteins of various size. They would have a number of scientific applications and also a medical application – delivery of treatment straight to cells that need it.

A normally dangerous virus – the hepatitis B virus – can become such a container. Viruses naturally are already containers, but containers of their own DNA. Thus they have the potential to be redesigned into protein or small molecule carriers. A number or viruses have... (More)
Molecular nano-carrier of proteins

Nature is using proteins to create an unfathomably rich variety of structures and functions. Some of them can be ‘’stolen’’ and adjust them for purposes and needs of science and humanity. One such function is a need for nanostructure that could contain enclose molecules and proteins of various size. They would have a number of scientific applications and also a medical application – delivery of treatment straight to cells that need it.

A normally dangerous virus – the hepatitis B virus – can become such a container. Viruses naturally are already containers, but containers of their own DNA. Thus they have the potential to be redesigned into protein or small molecule carriers. A number or viruses have already been demonstrated to fit this purpose, including HBV. However, this inclusion can be specific or non-specific. No specific examples with HBV have been performed yet. Therefore the HBV capsid is a perfect target to introduce specific binding capability into.

Viruses are complex and interesting structures, but is possible to multiply only the parts that are of interest for scientists. In this case the self-assembing capsid protein of HBV is produced in the most common organism used for such purpose – bacterium E.coli. Capsids are the sphere shaped, container like core structure of the virus. They can selfassemble from identical building blocks, the dimers (red in image). The part of the dimer, that faces the inside of spherical structure, the capsid, can be modified (red+blue in image) to bind a certain target (purple and green in image). If the building blocks are mixed with cargo they associate and build a capsid at the same time. Thus the cargo occurs in the inside of the complete capsid.

In this thesis, this capsid protein is purified from all other proteins of E.coli by method called size exclusion chromatography. The purified capsid protein is crystallized. The crystals are to be used to determine the three-dimensional structure of this protein, to know if all modified capsid parts look the same. The ability of modified dimers to bind the target is also shown experimentally. In addition, conditions for performing the assembly reaction are optimizied.

Advisor: Ingemar André
Master´s Degree Project 45 credits in Molecular Biology 2015
Department of Biochemistry and Structural Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Tupiņa, Dagnija
supervisor
organization
course
MOBN01 20151
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
7761633
date added to LUP
2015-08-14 11:01:01
date last changed
2016-04-18 13:38:44
@misc{7761633,
  abstract     = {Nanocarriers and nanoreactors are often derived from viral capsids. The capsid of HBV is a particularly attractive self-assembling model, fit for conversion into protein carrier. In this thesis modified HBV capsid is expressed in E.coli and purified by size exclusion chromatography. Nanocarrier system is based on principle of specific binding between the modified capsid building block and protein cargo. Binding of cargo and modified capsid building block is determined by fluorescence spectroscopy and binding constant is determined. Crystallization screen is performed to solve the structure and determine the position of the added tag. This will evaluate the possibility of seeing the structure of the cargo as well. In addition, self-assembly conditions are explored to potentially improve the encapsidation.},
  author       = {Tupiņa, Dagnija},
  language     = {eng},
  note         = {Student Paper},
  title        = {Purification, crystallization and characterization of modified HBV capsid},
  year         = {2015},
}