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Recombinant expression of the cowpea chlorotic mottle virus

Willas, Amanda LU (2019) KEMK03 20191
Department of Chemistry
Abstract
All viruses are composed of both genetic material and a capsid. The capsid is composed by identical protein subunits and the purpose is protection of the genetic material, in this case RNA. This project is about cowpea chlorotic mottle virus, also called CCMV, which is a plant virus and the disassembly and reassembly mechanisms of the capsid are investigated to understand how the capsid proteins associate to create capsids. These mechanisms are suitable to use as a model system to study protein self-assembly.
The capsid is able to disassembly and removes the genetic material. The capsid proteins can then assembly to an empty capsid without any genetic material.
The goal with this project is to develop a method to recombinant express... (More)
All viruses are composed of both genetic material and a capsid. The capsid is composed by identical protein subunits and the purpose is protection of the genetic material, in this case RNA. This project is about cowpea chlorotic mottle virus, also called CCMV, which is a plant virus and the disassembly and reassembly mechanisms of the capsid are investigated to understand how the capsid proteins associate to create capsids. These mechanisms are suitable to use as a model system to study protein self-assembly.
The capsid is able to disassembly and removes the genetic material. The capsid proteins can then assembly to an empty capsid without any genetic material.
The goal with this project is to develop a method to recombinant express CCMV and isolate dimers that are appropriate for assembly.
Previous research has tried to take advantages of a binding his-tag to facilitate the purification. These experiments have been unsuccessful. For that reason, this project doesn´t use a his-tag.
Plasmids containing the gene of interest were recombinant expressed in E.coli and purified with ammonium sulphate precipitation followed by gel filtration. Dynamic light scattering and mass spectrometry were performed on fractions from the gel filtration.
The solution conditions were changed to control assembly of capsid proteins. Dimers were isolated and experiments with a 50/50 mix of dimers and assembly buffer with different pH and incubation times were performed to create capsids. A 50/50 mix of dimers and assembly buffer with pH 4.5 and incubation for 30 minutes probably created capsids.
After the expression was the protein found in the soluble part of the clarified lysate. Previous research has found the protein in the inclusion bodies.
Further experiments should try to optimize the purification and also the solution conditions. The assembly-competent CCMV dimers can be recombinant expressed and isolated and thus useful in further biophysical experiments. (Less)
Popular Abstract (Swedish)
Virus är icke levande organismer som infekterar levande celler och förökar sig med hjälp av cellernas maskineri. De kan infektera alla typer av liv och de är totalt beroende av en värdcell. Alla virus innehåller genetiskt material samt en så kallad viruskapsid. Denna kapsid kan ha olika struktur och består av identiska proteinenheter. Kapsidens uppgift är att skydda det genetiska materialet. Detta projekt handlar om cowpea chlorotic mottle virus, även kallat CCMV, som är ett växtvirus med ikosaedrisk struktur vars associerings- och disassocieringsmekanismer fungerar som ett lämpligt modellsystem för att studera hur olika proteinenheter sätts ihop för att skapa en kapsid. Detta är ett passande modellsystem för att kapsiden kan disassociera... (More)
Virus är icke levande organismer som infekterar levande celler och förökar sig med hjälp av cellernas maskineri. De kan infektera alla typer av liv och de är totalt beroende av en värdcell. Alla virus innehåller genetiskt material samt en så kallad viruskapsid. Denna kapsid kan ha olika struktur och består av identiska proteinenheter. Kapsidens uppgift är att skydda det genetiska materialet. Detta projekt handlar om cowpea chlorotic mottle virus, även kallat CCMV, som är ett växtvirus med ikosaedrisk struktur vars associerings- och disassocieringsmekanismer fungerar som ett lämpligt modellsystem för att studera hur olika proteinenheter sätts ihop för att skapa en kapsid. Detta är ett passande modellsystem för att kapsiden kan disassociera och avlägsna det genetiska materialet för att sedan under specifika förhållanden associera tillbaka till den ursprungliga kapsiden.
Projektet har gått ut på att uttrycka CCMV genom rekombinant protein produktion. Proteinuttryck görs ofta i bakteriesystem eftersom bakterier både är lätta att odla och de växer snabbt. Dessutom producerar de ofta höga utbyten av rekombinant protein.
Escherichia coli, mer känd som E.coli, är en bakterieart och i detta projekt utnyttjas två olika slags E.coli celler.
Under projektet isolerades proteinenheter och det undersöktes under vilka förhållande som dessa proteinenheter associerar och skapar kapsider. Experimenten visade att med en associeringsbuffert och surt pH kunde troligen kapsider skapas.
Sammanfattningsvis är målet med projektet alltså att utveckla en metod för att rekombinant uttrycka CCMV följt av isolering av associeringskompententa dimerer. (Less)
Please use this url to cite or link to this publication:
author
Willas, Amanda LU
supervisor
organization
course
KEMK03 20191
year
type
M2 - Bachelor Degree
subject
keywords
Biokemi, Biochemistry, Viruskapsid, Viruscaspid, CCMV
language
English
id
8973986
date added to LUP
2019-07-04 13:31:20
date last changed
2019-07-04 13:31:20
@misc{8973986,
  abstract     = {{All viruses are composed of both genetic material and a capsid. The capsid is composed by identical protein subunits and the purpose is protection of the genetic material, in this case RNA. This project is about cowpea chlorotic mottle virus, also called CCMV, which is a plant virus and the disassembly and reassembly mechanisms of the capsid are investigated to understand how the capsid proteins associate to create capsids. These mechanisms are suitable to use as a model system to study protein self-assembly. 
The capsid is able to disassembly and removes the genetic material. The capsid proteins can then assembly to an empty capsid without any genetic material. 
The goal with this project is to develop a method to recombinant express CCMV and isolate dimers that are appropriate for assembly.
Previous research has tried to take advantages of a binding his-tag to facilitate the purification. These experiments have been unsuccessful. For that reason, this project doesn´t use a his-tag. 
Plasmids containing the gene of interest were recombinant expressed in E.coli and purified with ammonium sulphate precipitation followed by gel filtration. Dynamic light scattering and mass spectrometry were performed on fractions from the gel filtration. 
The solution conditions were changed to control assembly of capsid proteins. Dimers were isolated and experiments with a 50/50 mix of dimers and assembly buffer with different pH and incubation times were performed to create capsids. A 50/50 mix of dimers and assembly buffer with pH 4.5 and incubation for 30 minutes probably created capsids. 
After the expression was the protein found in the soluble part of the clarified lysate. Previous research has found the protein in the inclusion bodies. 
Further experiments should try to optimize the purification and also the solution conditions. The assembly-competent CCMV dimers can be recombinant expressed and isolated and thus useful in further biophysical experiments.}},
  author       = {{Willas, Amanda}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Recombinant expression of the cowpea chlorotic mottle virus}},
  year         = {{2019}},
}