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Purification of the Hylobius abietis TRPA1 ion channel

Lishöj, Vilda LU (2025) KEML10 20251
Department of Chemistry
Abstract
Hylobius abietis is a common pest in the forestry industry, damaging countless of conifer
seedlings each year. By solving the structure of the nociceptor, HaTRPA1, a repellent could
be developed to replace environmentally dangerous pesticides. To solve the structure, a large
amount needs to be purified. Previous problems plaguing previous studies include low protein
yield and contamination from Complex 1, associated with the electron transport chain.
Changes to already existing protocol were performed to optimize purification. A gradient use
of imidazole determined a better imidazole concentration at 60 mM for binding during
forward IMAC, to exclude Complex 1. A novel freezing method for increasing the effect of
solubilisation was... (More)
Hylobius abietis is a common pest in the forestry industry, damaging countless of conifer
seedlings each year. By solving the structure of the nociceptor, HaTRPA1, a repellent could
be developed to replace environmentally dangerous pesticides. To solve the structure, a large
amount needs to be purified. Previous problems plaguing previous studies include low protein
yield and contamination from Complex 1, associated with the electron transport chain.
Changes to already existing protocol were performed to optimize purification. A gradient use
of imidazole determined a better imidazole concentration at 60 mM for binding during
forward IMAC, to exclude Complex 1. A novel freezing method for increasing the effect of
solubilisation was investigated but showed no greater difference from normal solubilisation
methods. Optimisation of TEV cleavage was attempted with poorer methods for protein
concentration determination being the root cause of insufficient cleavage. An examination of
the effects on yield during forward IMAC, based on sample volume was attempted. However,
due to recent loss of binding on the column, this proved inconclusive. Further examination of
an alternative protein concentration determination would be beneficial along with increasing
protein stability to prevent aggregation. (Less)
Popular Abstract
The pungent, strong taste of mustard and wasabi comes from an interaction between a
chemical compound and a protein called TRPA1. In humans it generates this sensation and
reacts to other irritants, while in insects it can invoke a repellent behaviour. By studying the
protein’s structure in insects, a chemical can be developed which can be used as repellent in
place of insecticides.
Hylobius abietis, better known as the pine weevil, is one of these insects that present a major
challenge to the forestry industry, by destroying planted seedlings. To determine the structure
of TRPA1, a large amount of protein needs to be purified. This study concerns optimising the
already set protocol for purification. The protein is fitted with a... (More)
The pungent, strong taste of mustard and wasabi comes from an interaction between a
chemical compound and a protein called TRPA1. In humans it generates this sensation and
reacts to other irritants, while in insects it can invoke a repellent behaviour. By studying the
protein’s structure in insects, a chemical can be developed which can be used as repellent in
place of insecticides.
Hylobius abietis, better known as the pine weevil, is one of these insects that present a major
challenge to the forestry industry, by destroying planted seedlings. To determine the structure
of TRPA1, a large amount of protein needs to be purified. This study concerns optimising the
already set protocol for purification. The protein is fitted with a fluorescent marker to
differentiate it from sample contaminants, which is later removed from the structure. This step
proved quite problematic during the various purification schemes and was the central focus of
the experiments. Other minor optimisations were also examined. (Less)
Please use this url to cite or link to this publication:
author
Lishöj, Vilda LU
supervisor
organization
course
KEML10 20251
year
type
M2 - Bachelor Degree
subject
keywords
TRPA1, Purification, TEV, Volume, Biochemistry
language
English
id
9192569
date added to LUP
2025-06-05 09:34:56
date last changed
2025-06-05 09:34:56
@misc{9192569,
  abstract     = {{Hylobius abietis is a common pest in the forestry industry, damaging countless of conifer
seedlings each year. By solving the structure of the nociceptor, HaTRPA1, a repellent could
be developed to replace environmentally dangerous pesticides. To solve the structure, a large
amount needs to be purified. Previous problems plaguing previous studies include low protein
yield and contamination from Complex 1, associated with the electron transport chain.
Changes to already existing protocol were performed to optimize purification. A gradient use
of imidazole determined a better imidazole concentration at 60 mM for binding during
forward IMAC, to exclude Complex 1. A novel freezing method for increasing the effect of
solubilisation was investigated but showed no greater difference from normal solubilisation
methods. Optimisation of TEV cleavage was attempted with poorer methods for protein
concentration determination being the root cause of insufficient cleavage. An examination of
the effects on yield during forward IMAC, based on sample volume was attempted. However,
due to recent loss of binding on the column, this proved inconclusive. Further examination of
an alternative protein concentration determination would be beneficial along with increasing
protein stability to prevent aggregation.}},
  author       = {{Lishöj, Vilda}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Purification of the Hylobius abietis TRPA1 ion channel}},
  year         = {{2025}},
}