Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure
Alzheimer’s Disease, also called AD, is a horrible, degenerative brain disease that more than 35 million people over the world have. Today, there is no cure for this disease, only treatments that are temporarily relieving the symptoms. The two proteins that is thought to be the main cause of AD is a...
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Linköpings universitet, Institutionen för fysik, kemi och biologi
2020
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ftlinkoepinguniv:oai:DiVA.org:liu-167167 2023-05-15T14:55:22+02:00 Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure Fredén, Linnéa 2020 application/pdf http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167167 eng eng Linköpings universitet, Institutionen för fysik, kemi och biologi http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167167 info:eu-repo/semantics/openAccess Alzheimer’s Disease (AD) Amyloids Aβ fusion protein mNeonGreen mNG-Aβ1-42 Crystallization mouse brain tissue human brain tissue amyloid fluorescent probes Structural Biology Strukturbiologi Student thesis info:eu-repo/semantics/bachelorThesis text 2020 ftlinkoepinguniv 2022-05-01T08:22:55Z Alzheimer’s Disease, also called AD, is a horrible, degenerative brain disease that more than 35 million people over the world have. Today, there is no cure for this disease, only treatments that are temporarily relieving the symptoms. The two proteins that is thought to be the main cause of AD is amyloid β (Aβ) and tau. Previously, people have tried studying Aβ in vivo using green fluorescent protein fusion together with Aβ. However, this is difficult since the aggregation of Aβ will lead to loss of fluorescence. This study aimed to crystallize the fusion protein mNG-A β1-42 and to investigate its properties as a molecular fluorescent Aβ-amyloid specific probe. Dynamic light scattering (DLS) was used to confirm that the majority of the protein was not in the form of soluble aggregates. The DLS experiments were followed by several rounds of crystallization trials. Initial screening and the subsequent narrowing down of potential conditions where mNG-Aβ1-42 could form crystals. Several staining experiments were conducted as well, including staining brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. The DLS experiments showed that the protein used in the crystallization experiments mostly consisted of molecular particles of the same radius. However, there was clear evidence of some larger species present that could have been a potential problem for crystallization. Crystallization experiments suggested that PEG 8000 was the most promising precipitant amongst other conditions identified for crystallization of mNG-Aβ1-42. However, the study was ultimately unsuccessful in developing crystals of sufficient high quality for diffraction studies to commence. The staining experiments demonstrated that mNG-Aβ1-42 could bind both by itself and with another amyloid probe, Congo red, and with antibodies in brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. In conclusion, several characteristics of mNG-Aβ1-42 were revealed in this study. Bachelor Thesis Arctic LIU - Linköping University: Publications (DiVA) Arctic |
institution |
Open Polar |
collection |
LIU - Linköping University: Publications (DiVA) |
op_collection_id |
ftlinkoepinguniv |
language |
English |
topic |
Alzheimer’s Disease (AD) Amyloids Aβ fusion protein mNeonGreen mNG-Aβ1-42 Crystallization mouse brain tissue human brain tissue amyloid fluorescent probes Structural Biology Strukturbiologi |
spellingShingle |
Alzheimer’s Disease (AD) Amyloids Aβ fusion protein mNeonGreen mNG-Aβ1-42 Crystallization mouse brain tissue human brain tissue amyloid fluorescent probes Structural Biology Strukturbiologi Fredén, Linnéa Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure |
topic_facet |
Alzheimer’s Disease (AD) Amyloids Aβ fusion protein mNeonGreen mNG-Aβ1-42 Crystallization mouse brain tissue human brain tissue amyloid fluorescent probes Structural Biology Strukturbiologi |
description |
Alzheimer’s Disease, also called AD, is a horrible, degenerative brain disease that more than 35 million people over the world have. Today, there is no cure for this disease, only treatments that are temporarily relieving the symptoms. The two proteins that is thought to be the main cause of AD is amyloid β (Aβ) and tau. Previously, people have tried studying Aβ in vivo using green fluorescent protein fusion together with Aβ. However, this is difficult since the aggregation of Aβ will lead to loss of fluorescence. This study aimed to crystallize the fusion protein mNG-A β1-42 and to investigate its properties as a molecular fluorescent Aβ-amyloid specific probe. Dynamic light scattering (DLS) was used to confirm that the majority of the protein was not in the form of soluble aggregates. The DLS experiments were followed by several rounds of crystallization trials. Initial screening and the subsequent narrowing down of potential conditions where mNG-Aβ1-42 could form crystals. Several staining experiments were conducted as well, including staining brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. The DLS experiments showed that the protein used in the crystallization experiments mostly consisted of molecular particles of the same radius. However, there was clear evidence of some larger species present that could have been a potential problem for crystallization. Crystallization experiments suggested that PEG 8000 was the most promising precipitant amongst other conditions identified for crystallization of mNG-Aβ1-42. However, the study was ultimately unsuccessful in developing crystals of sufficient high quality for diffraction studies to commence. The staining experiments demonstrated that mNG-Aβ1-42 could bind both by itself and with another amyloid probe, Congo red, and with antibodies in brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. In conclusion, several characteristics of mNG-Aβ1-42 were revealed in this study. |
format |
Bachelor Thesis |
author |
Fredén, Linnéa |
author_facet |
Fredén, Linnéa |
author_sort |
Fredén, Linnéa |
title |
Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure |
title_short |
Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure |
title_full |
Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure |
title_fullStr |
Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure |
title_full_unstemmed |
Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure |
title_sort |
characterization of the fusion protein mng-aβ1-42 as a fluorescence reporter probe for amyloid structure |
publisher |
Linköpings universitet, Institutionen för fysik, kemi och biologi |
publishDate |
2020 |
url |
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167167 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_relation |
http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-167167 |
op_rights |
info:eu-repo/semantics/openAccess |
_version_ |
1766327164360196096 |