Team:Wageningen UR/ObtainingthePoleroVLP

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== Introduction ==
== Introduction ==
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The Polero (potato leaf roll virus) virus coat proteins can be used as building blocks to form VLP’s, which has never been done before in E. coli. Therefore we will focus on the use of Polero virus coat proteins to make self-assembling VLP’s. If this step succeeds, further modifications will be done. After formation of the VLP’s, the spike will be presented on the outside, so we will mainly focus on modifying the spike on the outside. We will start with small peptides and then try to add bigger ligands; finally we will see where the boundary lies of how big the proteins can be that are added to the end of the spike.
 
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'''Aim''': Our primary aim is to get self-assembling VLP’s from the Polero virus in E. coli. If we succeed, we can attempt to modify the spike on the outside.
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== Isolation and BioBricking of the potato leafroll virus (PLRV) coat protein. ==
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The Polero (potato leaf roll virus, PLRV) virus coat proteins can be used as building blocks to form VLP’s. PLRV is a positive sense RNA virus (group IV). Viruses from this group have their genome directly utilized as if it were mRNA. Ribosomes from the host cell translate their genome into a protein, with RNA-dependent RNA polymerase as one of it. This means that to isolate the coat protein gene we need to isolate the RNA of infected potato plants which would include the viral RNA.
 +
 
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After obtaining the coat protein gene of PLRV, the PCR product can be sent to be sequenced. The sequence of our isolate together with other isolates will give us information about the conserved regions in the gene. Alignment experiments can be performed, also together with data of other members of the Luteoviridae.
 +
The PLRV coat protein can thereafter be ‘’BioBricked’’. This brick can be expressed in Escherichia coli and produced monomers can be isolated and purified.
 +
 
 +
In nature, the stopcodon of the coat protein is sometimes ‘’missed’’, which results in a 70kDa readthrough product. This is considerably larger than the 23kDa coat protein on its own. When the coat protein as well as the coat protein plus readthrough are assembled together to form the virus capsid, spikes are formed on the capsid. Virus like particles (VLPs) are being formed either with or without readtrough proteins. The expression of coat protein monomers, needed for VLP formation, has never been done in E.coli. If this step succeeds, further modifications on the VLP’s will be done.
 +
 
 +
 
 +
'''Aim''': Our primary aim is to get self-assembling VLP’s from PLRV in E. coli. If we succeed, we can attempt to modify the spike on the outside.
 +
 
 +
Bricking coat protein gene of potato leafroll virus (PRLV) from Wageningen potatoes.
 +
 +
 
 +
'''Progress''':
 +
 
 +
RNA isolation of potato leaf tissue
 +
 
 +
Reverse transcriptase reaction to obtain cDNA
 +
 
 +
PCR on cDNA using coat protein primers
 +
 
 +
Addition of Prefix and Suffix to CP genes
 +
 
 +
 

Revision as of 13:01, 27 July 2012


Introduction

Isolation and BioBricking of the potato leafroll virus (PLRV) coat protein.

The Polero (potato leaf roll virus, PLRV) virus coat proteins can be used as building blocks to form VLP’s. PLRV is a positive sense RNA virus (group IV). Viruses from this group have their genome directly utilized as if it were mRNA. Ribosomes from the host cell translate their genome into a protein, with RNA-dependent RNA polymerase as one of it. This means that to isolate the coat protein gene we need to isolate the RNA of infected potato plants which would include the viral RNA.

After obtaining the coat protein gene of PLRV, the PCR product can be sent to be sequenced. The sequence of our isolate together with other isolates will give us information about the conserved regions in the gene. Alignment experiments can be performed, also together with data of other members of the Luteoviridae. The PLRV coat protein can thereafter be ‘’BioBricked’’. This brick can be expressed in Escherichia coli and produced monomers can be isolated and purified.

In nature, the stopcodon of the coat protein is sometimes ‘’missed’’, which results in a 70kDa readthrough product. This is considerably larger than the 23kDa coat protein on its own. When the coat protein as well as the coat protein plus readthrough are assembled together to form the virus capsid, spikes are formed on the capsid. Virus like particles (VLPs) are being formed either with or without readtrough proteins. The expression of coat protein monomers, needed for VLP formation, has never been done in E.coli. If this step succeeds, further modifications on the VLP’s will be done.


Aim: Our primary aim is to get self-assembling VLP’s from PLRV in E. coli. If we succeed, we can attempt to modify the spike on the outside.

Bricking coat protein gene of potato leafroll virus (PRLV) from Wageningen potatoes.


Progress:

RNA isolation of potato leaf tissue

Reverse transcriptase reaction to obtain cDNA

PCR on cDNA using coat protein primers

Addition of Prefix and Suffix to CP genes

 

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