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Team:Wageningen UR/InsideModification
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=== Introduction === | === Introduction === | ||
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Since virus-like particles (VLPs) lack genetic content, they enclose an empty space. This space can be filled with all sorts of proteins such as antibiotics, hormones, and all sorts of farmaceuticals. Modifications on the inside of the VLPs opens up more options. One of the modifications we pursue is adding a coil to the protein subunits, at any location that is exposed on the inside of the VLP. In our case, this is a fusion to either the N-terminal or the C-terminal. The coils will facilitate stronger attachment to the substance which needs to stick to the VLP's interior. | Since virus-like particles (VLPs) lack genetic content, they enclose an empty space. This space can be filled with all sorts of proteins such as antibiotics, hormones, and all sorts of farmaceuticals. Modifications on the inside of the VLPs opens up more options. One of the modifications we pursue is adding a coil to the protein subunits, at any location that is exposed on the inside of the VLP. In our case, this is a fusion to either the N-terminal or the C-terminal. The coils will facilitate stronger attachment to the substance which needs to stick to the VLP's interior. | ||
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'''Aim:''' Construct a Virus-Like Particle that has coils exposed on the inside. | '''Aim:''' Construct a Virus-Like Particle that has coils exposed on the inside. | ||
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Another way to open up more options for loading our VLPs, is to make a change in the interior's charge. Normally, the interior of a VLP is positively charged, facilitating interaction with negatively charged DNA/RNA molecules. By changing the amino acid sequence that is located on the interior of a VLP, the positive charge can be changed into a negative charge. A VLP with a negatively charged interior can be loaded with all sorts of positively charged molecules, such as polymeres, metals, etc. | Another way to open up more options for loading our VLPs, is to make a change in the interior's charge. Normally, the interior of a VLP is positively charged, facilitating interaction with negatively charged DNA/RNA molecules. By changing the amino acid sequence that is located on the interior of a VLP, the positive charge can be changed into a negative charge. A VLP with a negatively charged interior can be loaded with all sorts of positively charged molecules, such as polymeres, metals, etc. | ||
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'''Aim:''' Construct a Virus-Like Particle that has a negatively charged interior. | '''Aim:''' Construct a Virus-Like Particle that has a negatively charged interior. | ||
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By modifying the CCMV VLP, we managed to add a negative charge to the interior, enabling us to load metals to the inside. | By modifying the CCMV VLP, we managed to add a negative charge to the interior, enabling us to load metals to the inside. | ||
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=== CCMV === | === CCMV === | ||
| - | + | The Cow Chlorotic Mottle Virus (CCMV) VLP is one of the best candidates for drug delivery, since it is tolerant of high temperatures and stable in a wide pH range. Next to that, the wild-type CCMV VLP is not specific for mammalian cells in vivo. Because the CCMv VLP is such a good candidate, we decided to place coils on the interior. Since the N-terminus of the CCMV VLP subunits is folded to the inside, this was the logic place to attach the coil to. | |
| + | To get the most out of the CCMV VLP, the interior was given a negative charge. As a proof of concept, the CCMV VLPs with a negative interior were loaded with iron ions and monitored using electron microscopy. While the wild-type CCMV VLP can not be loaded with positively charged substances, this modified version can. | ||
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| + | '''Results:''' | ||
| + | Attachment of a coil to the N-terminus of the CCMV VLP subunits was carried out in 4 subsequent PCR steps. Unluckily, the primer of the first step was not designed correctly, resulting in a frameshift over the entire VLP, making | ||
Revision as of 14:29, 20 September 2012
Contents |
Inside Modifications
Introduction
Since virus-like particles (VLPs) lack genetic content, they enclose an empty space. This space can be filled with all sorts of proteins such as antibiotics, hormones, and all sorts of farmaceuticals. Modifications on the inside of the VLPs opens up more options. One of the modifications we pursue is adding a coil to the protein subunits, at any location that is exposed on the inside of the VLP. In our case, this is a fusion to either the N-terminal or the C-terminal. The coils will facilitate stronger attachment to the substance which needs to stick to the VLP's interior.
Aim: Construct a Virus-Like Particle that has coils exposed on the inside.
Another way to open up more options for loading our VLPs, is to make a change in the interior's charge. Normally, the interior of a VLP is positively charged, facilitating interaction with negatively charged DNA/RNA molecules. By changing the amino acid sequence that is located on the interior of a VLP, the positive charge can be changed into a negative charge. A VLP with a negatively charged interior can be loaded with all sorts of positively charged molecules, such as polymeres, metals, etc.
Aim: Construct a Virus-Like Particle that has a negatively charged interior.
By modifying the CCMV VLP, we managed to add a negative charge to the interior, enabling us to load metals to the inside.
CCMV
The Cow Chlorotic Mottle Virus (CCMV) VLP is one of the best candidates for drug delivery, since it is tolerant of high temperatures and stable in a wide pH range. Next to that, the wild-type CCMV VLP is not specific for mammalian cells in vivo. Because the CCMv VLP is such a good candidate, we decided to place coils on the interior. Since the N-terminus of the CCMV VLP subunits is folded to the inside, this was the logic place to attach the coil to.
To get the most out of the CCMV VLP, the interior was given a negative charge. As a proof of concept, the CCMV VLPs with a negative interior were loaded with iron ions and monitored using electron microscopy. While the wild-type CCMV VLP can not be loaded with positively charged substances, this modified version can.
Results:
Attachment of a coil to the N-terminus of the CCMV VLP subunits was carried out in 4 subsequent PCR steps. Unluckily, the primer of the first step was not designed correctly, resulting in a frameshift over the entire VLP, making
Hepatitis B
Results:
