Screening for interaction domain in vault nanocapsules.

Protein nanocapsules are gaining popularity as therapeutic delivery vehicle particularly due to the potential of controlled release kinetics, particularly interesting is pH-controlled molecular release from the protein nanocapsule to release the content in the endosomes. Vault is a naturally assembl...

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Main Author: Loo, Jonathan Yun Haw.
Other Authors: Lim Sierin
Format: Final Year Project
Language:English
Published: 2013
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Online Access:http://hdl.handle.net/10356/51094
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-510942023-03-03T15:40:38Z Screening for interaction domain in vault nanocapsules. Loo, Jonathan Yun Haw. Lim Sierin School of Chemical and Biomedical Engineering DRNTU::Engineering::Bioengineering Protein nanocapsules are gaining popularity as therapeutic delivery vehicle particularly due to the potential of controlled release kinetics, particularly interesting is pH-controlled molecular release from the protein nanocapsule to release the content in the endosomes. Vault is a naturally assembled hollow protein nanocapsule (75 x 42 x 41 nm3) with a large inner cavity and is mostly composed of major vault proteins (MVP). MVP interacts with C-terminus of vault poly(ADP-ribose)-polymerase (VPARP), namely MVP-minimal interaction domain (mINT) at a domain near the waist of the vault complex in the inner cavity. This interaction between MVP and mINT is important as researches have shown that the mINT may serves as a protein shuttle to load proteins and nanoparticles into the inner cavity of the vault complex as well as the fact that the interaction site is near the waist of the nanocapsule, where the vault opens up under certain conditions. Modifying the interaction site will allow the engineering of non-native pH-dependent interaction functionality onto the vault nanocapsule to open and release its content at specific pH. This project attempted to identify specific domains that are involved in the interactions between MVP and mINT by studying the affinity between MVP domain 3-4 and 4-5 with the mINT. We constructed four plasmids in E. coli expression vector pET-42 b(+) and pET-11a encoding MVP domain 3-4 and 4-5 and mCherry-mINT respectively. These genes were overexpressed to produce protein fractions for validation of interactions. Attempts to over-express GST-MVP45 failed and yielded only GST-tag proteins. Results from affinity test done by running mixture of GST-MVP34 and mCherry-mINT through size exclusion chromatography using Superdex 200 show no or insufficient binding between the two proteins. Bachelor of Engineering (Chemical and Biomolecular Engineering) 2013-01-08T04:48:51Z 2013-01-08T04:48:51Z 2012 2012 Final Year Project (FYP) http://hdl.handle.net/10356/51094 en Nanyang Technological University 65 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Engineering::Bioengineering
spellingShingle DRNTU::Engineering::Bioengineering
Loo, Jonathan Yun Haw.
Screening for interaction domain in vault nanocapsules.
description Protein nanocapsules are gaining popularity as therapeutic delivery vehicle particularly due to the potential of controlled release kinetics, particularly interesting is pH-controlled molecular release from the protein nanocapsule to release the content in the endosomes. Vault is a naturally assembled hollow protein nanocapsule (75 x 42 x 41 nm3) with a large inner cavity and is mostly composed of major vault proteins (MVP). MVP interacts with C-terminus of vault poly(ADP-ribose)-polymerase (VPARP), namely MVP-minimal interaction domain (mINT) at a domain near the waist of the vault complex in the inner cavity. This interaction between MVP and mINT is important as researches have shown that the mINT may serves as a protein shuttle to load proteins and nanoparticles into the inner cavity of the vault complex as well as the fact that the interaction site is near the waist of the nanocapsule, where the vault opens up under certain conditions. Modifying the interaction site will allow the engineering of non-native pH-dependent interaction functionality onto the vault nanocapsule to open and release its content at specific pH. This project attempted to identify specific domains that are involved in the interactions between MVP and mINT by studying the affinity between MVP domain 3-4 and 4-5 with the mINT. We constructed four plasmids in E. coli expression vector pET-42 b(+) and pET-11a encoding MVP domain 3-4 and 4-5 and mCherry-mINT respectively. These genes were overexpressed to produce protein fractions for validation of interactions. Attempts to over-express GST-MVP45 failed and yielded only GST-tag proteins. Results from affinity test done by running mixture of GST-MVP34 and mCherry-mINT through size exclusion chromatography using Superdex 200 show no or insufficient binding between the two proteins.
author2 Lim Sierin
author_facet Lim Sierin
Loo, Jonathan Yun Haw.
format Final Year Project
author Loo, Jonathan Yun Haw.
author_sort Loo, Jonathan Yun Haw.
title Screening for interaction domain in vault nanocapsules.
title_short Screening for interaction domain in vault nanocapsules.
title_full Screening for interaction domain in vault nanocapsules.
title_fullStr Screening for interaction domain in vault nanocapsules.
title_full_unstemmed Screening for interaction domain in vault nanocapsules.
title_sort screening for interaction domain in vault nanocapsules.
publishDate 2013
url http://hdl.handle.net/10356/51094
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