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Team:Wageningen UR/OutsideModification
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Besides, there is only one insert needed, because the read-through does not occur with each translation. | Besides, there is only one insert needed, because the read-through does not occur with each translation. | ||
| - | '''Results''' | + | '''Results:''' |
Both viruses have been isolated from either nature or provided plasmids. We cloned both the full gene including read-through and the gene without read-through. The primers have been designed to elongate the different versions of the gene, adding the k-coil. [EM pending] | Both viruses have been isolated from either nature or provided plasmids. We cloned both the full gene including read-through and the gene without read-through. The primers have been designed to elongate the different versions of the gene, adding the k-coil. [EM pending] | ||
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There is one problem concerning the insertion of the k-coil in the external loop: The coil will be bended [link to in silico], while it is designed to be linear. This can be solved by inserting a protease specific site next to the coil. In this way, the coil can be linearized by cutting the protein after VLP formation. This exposes the coil in a linearized formation, allowing attachment of the ligand. | There is one problem concerning the insertion of the k-coil in the external loop: The coil will be bended [link to in silico], while it is designed to be linear. This can be solved by inserting a protease specific site next to the coil. In this way, the coil can be linearized by cutting the protein after VLP formation. This exposes the coil in a linearized formation, allowing attachment of the ligand. | ||
| - | '''Results''' | + | '''Results:''' |
Expression and formation of wild-type VLPs succeeded. Multiple ways to modify the loop were tried, but without success. | Expression and formation of wild-type VLPs succeeded. Multiple ways to modify the loop were tried, but without success. | ||
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[add Phyre2 image] | [add Phyre2 image] | ||
| - | '''Results''' | + | '''Results:''' |
The full construct of the delta 26 mutant was amplified and cloned. After sequencing and conformation of intended modifications, we found out that one of the many primers contained a minor mistake having major implications: a frameshift in the CCMV coat protein gene. However, this showed that the technique of extension-pcr worked, so the primers for TuYV should work as well. | The full construct of the delta 26 mutant was amplified and cloned. After sequencing and conformation of intended modifications, we found out that one of the many primers contained a minor mistake having major implications: a frameshift in the CCMV coat protein gene. However, this showed that the technique of extension-pcr worked, so the primers for TuYV should work as well. | ||
Revision as of 19:26, 19 September 2012
Contents |
Modifying the outside of a VLP
Introduction
The monomers of virus-like particles (VLPs) have been subject to many modifications of which some are aimed at changing the appearance of the particle. By changing the outside, the VLP acquires new properties which have been used mainly in vaccine development (ref 5epis and others). The modification we pursue is adding a coil to the protein subunits, at any location that is exposed on the outside of the VLP. This can be a fusion to a C or N-terminal, but a modification in a loop is possible as well. The goal of the coil is to serve as a docking site for ligands which are modified to contain a coil subunit. A coil on the outside plays a rather important role expanding the applications of VLPs. It forms the link between the VLP and a functional group, such as a ligand or antigen.
Aim: Construct a Virus-Like Particle that has the k-coil exposed on the outside
[schematic picture (from movie) with the coil on the outside]
PLRV and TuYV
The Potato Leaf Roll Virus (PLRV) and Turnip Yellows Virus (TuYV) have a very interesting feature. Part of the subunits is expressed with a read-through product which forms spikes-like structures on the outside of the VLP. The idea is to replace the spike by a k-coil. Because this part is not involved in formation of the VLP, it will not change the wild-type properties of the particle during formation. Besides, there is only one insert needed, because the read-through does not occur with each translation.
Results: Both viruses have been isolated from either nature or provided plasmids. We cloned both the full gene including read-through and the gene without read-through. The primers have been designed to elongate the different versions of the gene, adding the k-coil. [EM pending]
Hepatitis B
A loop exposed on the outside of Hepatitis B is known to accept modifications and for VLPs still [ref to research about inserts]. Formation is improved when a mixture is made from wild-type and modified subunits. There is one problem concerning the insertion of the k-coil in the external loop: The coil will be bended [link to in silico], while it is designed to be linear. This can be solved by inserting a protease specific site next to the coil. In this way, the coil can be linearized by cutting the protein after VLP formation. This exposes the coil in a linearized formation, allowing attachment of the ligand.
Results: Expression and formation of wild-type VLPs succeeded. Multiple ways to modify the loop were tried, but without success.
[make picture of loop before cutting, while cutting and after cutting with TEV]
CCMV
While Hepatitis B is modified multiple times and PLRV shows great opportunities to do so too, CCMV has not been modified on the outside yet. We have designed two possible modifications which might lead to the desired VLPs as described in the aim.
- Modification of an outside loop. This has shown to be possible with Hepatitis B, but the loops in CCMV seem to have more interactions
[add Phyre2 image of CCMV and circle loop3]
- Construct a mutant by deleting the first 26 amino acids, after which the k-coil is added. This might expose the k-coil on the outside of the VLP.
[add Phyre2 image]
Results: The full construct of the delta 26 mutant was amplified and cloned. After sequencing and conformation of intended modifications, we found out that one of the many primers contained a minor mistake having major implications: a frameshift in the CCMV coat protein gene. However, this showed that the technique of extension-pcr worked, so the primers for TuYV should work as well.
