Optimizing Expression and Purification of HaTRPA1
(2024) KEMR30 20241Department of Chemistry
- Abstract
- TRPA1, also known as the wasabi receptor, is a non-selective cation ion channel that plays an essential role in the detection of noxious substances and irritants. Hylobius abietis, the pine weevil, is a pest that causes substantial damage to the forestry industry. The structure of the pine weevils TRPA1 (HaTRPA1) is so far unknown. If the structure were to be determined, an agonist could be developed, which could be used as a repellent against the pine weevil. Membrane proteins are naturally expressed at lower levels and can be highly unstable in certain conditions. Heterologous expression and different protocols must often be attempted and evaluated to produce a high-yield, stable membrane protein sample. The aim of this study is to... (More)
- TRPA1, also known as the wasabi receptor, is a non-selective cation ion channel that plays an essential role in the detection of noxious substances and irritants. Hylobius abietis, the pine weevil, is a pest that causes substantial damage to the forestry industry. The structure of the pine weevils TRPA1 (HaTRPA1) is so far unknown. If the structure were to be determined, an agonist could be developed, which could be used as a repellent against the pine weevil. Membrane proteins are naturally expressed at lower levels and can be highly unstable in certain conditions. Heterologous expression and different protocols must often be attempted and evaluated to produce a high-yield, stable membrane protein sample. The aim of this study is to optimize and evaluate different protocols to express and purify HaTRPA1, including using a truncated version of HaTRPA1 and a cleavable green fluorescent protein tag.
HaTRPA1 was expressed through large-scale fermentation in Pichia pastoris. The cells were broken, and the membrane was isolated and washed. The protein was solubilized using Fos-choline 14, purified using Immobilized metal ion affinity chromatography (IMAC), cleavage with the tobacco etch virus protease, reverse IMAC, and separated using size exclusion chromatography. Steps throughout the expression and purification were evaluated using SDS-PAGE. The protocols were attempted for two constructs of HaTRPA1, the full-length construct and a truncated version labelled C2. The results showed that degradation was the major issue with the expression and purification of HaTRPA1. Both the full-length construct and the truncated C2 showed substantial degradation. A decrease in degradation was observed when the induction time during expression was reduced. Implementing a urea wash in the membrane preparation was beneficial in reducing the amount of degradation.
The conclusion of this paper was that there is little difference in stability between the two constructs of HaTRPA1. To prevent degradation, a shorter induction time should be implemented during fermentation. (Less) - Popular Abstract
- Have you ever put too much wasabi on a piece of sushi? The painful sensation you feel that causes instant regret is generated by the so-called Transient Receptor Potential Channel, subfamily A, member 1 (TRPA1). The protein TRPA1 plays an essential role in detecting irritants and noxious substances, like our dear friend wasabi. While wasabi may be harmless, there are many substances that can be highly damaging, and our bodies need a way to tell us to stay away from them. Therefore, once TRPA1 is activated by a noxious substance, a signal is sent to our nervous system to generate a painful sensation. While human TRPA1 has been the subject of many studies, it is also worth studying TRPA1 in other organisms.
Hylobius abietis, or the pine... (More) - Have you ever put too much wasabi on a piece of sushi? The painful sensation you feel that causes instant regret is generated by the so-called Transient Receptor Potential Channel, subfamily A, member 1 (TRPA1). The protein TRPA1 plays an essential role in detecting irritants and noxious substances, like our dear friend wasabi. While wasabi may be harmless, there are many substances that can be highly damaging, and our bodies need a way to tell us to stay away from them. Therefore, once TRPA1 is activated by a noxious substance, a signal is sent to our nervous system to generate a painful sensation. While human TRPA1 has been the subject of many studies, it is also worth studying TRPA1 in other organisms.
Hylobius abietis, or the pine weevil, is a major pest in the commercial forest industry. It eats the bark of seedlings causing them to die. The pine weevil is responsible for millions of euros in damages each year. Currently, the fight against the pine weevil has involved plenty of harmful insecticides, and with a greater focus on environmentally friendly forestry, there is an interest in other alternatives. If the 3D structure of H. abietis TRPA1 was resolved, it would be possible to develop an activator of TRPA1 that would trigger a similar response in the pine weevil as our overindulgence in wasabi caused.
This study attempted different conditions and protocols to produce a substantial amount of TRPA1 from the pine weevil for structural studies. Two different constructs were studied: the full-length construct and a truncated version. Both constructs of the protein were tagged with a green fluorescent protein to enable tracking of the protein throughout the purification and expression. The protein was expressed in the yeast species Pichia pastoris. Afterwards, the protein was solubilized and purified under different conditions to find the optimal protocol. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9159056
- author
- Hansson Wennersten, Wilhelm LU
- supervisor
- organization
- course
- KEMR30 20241
- year
- 2024
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Degradation, Fermentation, GFP, TRPA1, Biochemistry
- language
- English
- id
- 9159056
- date added to LUP
- 2024-06-11 10:47:04
- date last changed
- 2024-06-11 10:47:04
@misc{9159056,
abstract = {{TRPA1, also known as the wasabi receptor, is a non-selective cation ion channel that plays an essential role in the detection of noxious substances and irritants. Hylobius abietis, the pine weevil, is a pest that causes substantial damage to the forestry industry. The structure of the pine weevils TRPA1 (HaTRPA1) is so far unknown. If the structure were to be determined, an agonist could be developed, which could be used as a repellent against the pine weevil. Membrane proteins are naturally expressed at lower levels and can be highly unstable in certain conditions. Heterologous expression and different protocols must often be attempted and evaluated to produce a high-yield, stable membrane protein sample. The aim of this study is to optimize and evaluate different protocols to express and purify HaTRPA1, including using a truncated version of HaTRPA1 and a cleavable green fluorescent protein tag.
HaTRPA1 was expressed through large-scale fermentation in Pichia pastoris. The cells were broken, and the membrane was isolated and washed. The protein was solubilized using Fos-choline 14, purified using Immobilized metal ion affinity chromatography (IMAC), cleavage with the tobacco etch virus protease, reverse IMAC, and separated using size exclusion chromatography. Steps throughout the expression and purification were evaluated using SDS-PAGE. The protocols were attempted for two constructs of HaTRPA1, the full-length construct and a truncated version labelled C2. The results showed that degradation was the major issue with the expression and purification of HaTRPA1. Both the full-length construct and the truncated C2 showed substantial degradation. A decrease in degradation was observed when the induction time during expression was reduced. Implementing a urea wash in the membrane preparation was beneficial in reducing the amount of degradation.
The conclusion of this paper was that there is little difference in stability between the two constructs of HaTRPA1. To prevent degradation, a shorter induction time should be implemented during fermentation.}},
author = {{Hansson Wennersten, Wilhelm}},
language = {{eng}},
note = {{Student Paper}},
title = {{Optimizing Expression and Purification of HaTRPA1}},
year = {{2024}},
}