Utilization of -1 frameshifting to construct an expression reporter for capsid proteins
(2019) KEMK03 20191Department of Chemistry
- Abstract
- Virus capsids shows promise within a wide variety of applications. However, the current lack of understanding about their self-assembly limits their usefulness. A study of the amino acids relevant for their assembly would be of great use by giving insight to the mechanic behind it. Since the number of proteins involved in each capsid is so big, there is a need for a reporter system enabling easy assaying of the stability between different mutations of the protein. A system proposed allows simultaneous monitoring of both the production and stability of the capsid forming protein in vivo. The production monitoring part is created by linking a fluorescent gene to the protein gene, creating a fusion protein with a measurable signal. In this... (More)
- Virus capsids shows promise within a wide variety of applications. However, the current lack of understanding about their self-assembly limits their usefulness. A study of the amino acids relevant for their assembly would be of great use by giving insight to the mechanic behind it. Since the number of proteins involved in each capsid is so big, there is a need for a reporter system enabling easy assaying of the stability between different mutations of the protein. A system proposed allows simultaneous monitoring of both the production and stability of the capsid forming protein in vivo. The production monitoring part is created by linking a fluorescent gene to the protein gene, creating a fusion protein with a measurable signal. In this project a production monitoring system is created where only a small proportion of the translation leads to the fusion protein. This in order to minimize the destabilizing effect of the fluorophore on the protein’s capsid forming ability, while still allowing a signal that can be converted into amount of protein produced. This is achieved by digestion ligation where a frameshift sequence is placed between the two genes. The frameshift sequence directs how much of non-fusion to fusion protein is expressed during translation. (Less)
- Popular Abstract
- Viruses are the cause of a large variety of diseases, some currently cureless, like HIV, and some harmless but persistent, like the common cold. One of the things giving the viruses such perseverance is the protective protein coat, called capsid, that encapsulate their genomic material. This capsid packs, protects and delivers the genomic material to the cell intended for infection.
The capsids are complex and the proteins forming the capsids will spontaneously assemble into their capsid formation when in contact with each other. The mechanism behind this self-assembly is poorly understood. If knowledge about the driving forces behind this assembly could be widened this would be useful in cases where the virus eludes the body’s own... (More) - Viruses are the cause of a large variety of diseases, some currently cureless, like HIV, and some harmless but persistent, like the common cold. One of the things giving the viruses such perseverance is the protective protein coat, called capsid, that encapsulate their genomic material. This capsid packs, protects and delivers the genomic material to the cell intended for infection.
The capsids are complex and the proteins forming the capsids will spontaneously assemble into their capsid formation when in contact with each other. The mechanism behind this self-assembly is poorly understood. If knowledge about the driving forces behind this assembly could be widened this would be useful in cases where the virus eludes the body’s own immune system by hiding within capsids, as is the case with HIV. Virus capsids would be a good target for antiviral drugs seeing as the drugs existing today often targets the virus after it has entered the cell, harming the host cell in the process.
Furthermore, additional knowledge of the capsids would make it possible to manipulate and use them for other purposes. We could modify the protein to encapsulate molecules other than the virus’s genomic material and steer the cell targeting of the capsid. With this level of control very specific drug delivery could be obtained, for example if anticancer drugs could be delivered by targeting only the cancer cells, this would mean a much less harmful treatment than the alternatives currently available.
This is only a small fraction of the possibilities that extended knowledge could lead to. There is a large field of research being made with the purpose of utilizing the capsids unique properties for various purposes. There has been attempts to replicate this self-assembly found in nature, but so far none of the engineered capsids have come close to the sophisticated structures found in virus capsids.
In this group the aim is to build a library of knowledge about one specific virus capsid, the Hepatitis B virus capsid. This library is intended to expand the knowledge of capsid self-assembly by mapping which parts of the Hepatitis B virus’s capsid forming protein is relevant in its assembly. This is accomplished by making small changes in the protein and comparing the stability between the resulting capsids.
One of the problems when studying capsid formation is the large number of proteins that makes up each capsid. In the case of the Hepatitis B virus each capsid consists of either 180 or 240 proteins, this results in a large amount of data when studying these proteins. Therefore, this group is also working towards creating a system, allowing for fast and easy monitoring of these large quantities as they are produced within a cell. The principle of the system is to have one signal reporter, in the form of a fluorescent protein, that is related to the amount of protein the cell expresses and another signal reporter that will indicate how well the protein forms capsids.
In my project I focused on the production measuring part of this system. The easiest way to directly measure the production of a protein is to have the signal reporter directly linked to the protein. However, this alteration to the protein will most likely affect the capsid forming ability of the protein. For the production signal to be of use when compared to the stability signal this interference must be minimised. The system I created makes it so that only a small percentage of the protein expressed will be linked to a reporter, allowing for most of the protein to be unaffected. With this the production signal can still be used to calculate the amount of protein that contributes to the stability signal.
This project is one small step in the direction towards better understanding the mechanics behind capsid assembly. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8987446
- author
- Ahl, Erica LU
- supervisor
- organization
- course
- KEMK03 20191
- year
- 2019
- type
- M2 - Bachelor Degree
- subject
- keywords
- biochemistry, biokemi, Virus capsid, frameshifting
- language
- English
- id
- 8987446
- date added to LUP
- 2019-07-04 16:24:28
- date last changed
- 2019-07-04 16:24:28
@misc{8987446,
abstract = {{Virus capsids shows promise within a wide variety of applications. However, the current lack of understanding about their self-assembly limits their usefulness. A study of the amino acids relevant for their assembly would be of great use by giving insight to the mechanic behind it. Since the number of proteins involved in each capsid is so big, there is a need for a reporter system enabling easy assaying of the stability between different mutations of the protein. A system proposed allows simultaneous monitoring of both the production and stability of the capsid forming protein in vivo. The production monitoring part is created by linking a fluorescent gene to the protein gene, creating a fusion protein with a measurable signal. In this project a production monitoring system is created where only a small proportion of the translation leads to the fusion protein. This in order to minimize the destabilizing effect of the fluorophore on the protein’s capsid forming ability, while still allowing a signal that can be converted into amount of protein produced. This is achieved by digestion ligation where a frameshift sequence is placed between the two genes. The frameshift sequence directs how much of non-fusion to fusion protein is expressed during translation.}},
author = {{Ahl, Erica}},
language = {{eng}},
note = {{Student Paper}},
title = {{Utilization of -1 frameshifting to construct an expression reporter for capsid proteins}},
year = {{2019}},
}