Heterologous expression and purification of GFP tagged HaTRPA1 exploiting the yeast Pichia pastoris

Abstract

Transient Receptor Potential Ankyrin 1 (TRPA1) is a non-selective cation channel that plays a significant role in pain, itch, and inflammatory conditions, and its activation is mediated by various electrophilic compounds and small molecules like menthol and camphor. Understanding the mechanism of activation and deactivation of TRPA1 is crucial for developing treatments for these conditions.
This study focuses on TRPA1 from Hylobius abietis (HaTRPA1), in other words a pine weevil which is an insect that causes coniferous tree mortality by eating the bark. The insights gained from this study could potentially aid in the development of a pine weevil repellent, thus preserving the trees. Additionally, understanding the structure and function of HaTRPA1 may simplify further studies on this type of protein.
The investigation of HaTRPA1 is hindered by its intrinsic difficulties, including low expression levels and solubilization issues. This study aimed to surmount these challenges by identifying the optimal expression clone, selecting a suitable detergent for effective protein solubilization, and employing various chromatographic methods, such as immobilized metal affinity chromatography (IMAC), reversed IMAC, and size exclusion chromatography (SEC), for protein purification.
After the selection and purification of the most favorable clone, fos-choline-14 was identified as the most effective detergent, addressing both solubilization and purification challenges associated with HaTRPA1. However, the protein's stability was assessed using amphipol PMAL-C8 and SEC methods, revealing inadequacies that call for further improvements in the purification process and the identification of more compatible detergents.
To tackle the specific obstacles related to HaTRPA1, this study harnessed the advantages offered by GFP-tagging. The incorporation of the GFP-tag enabled the visualization and tracking of HaTRPA1 within living cells, allowing to monitor its localization and dynamics, despite its low expression levels. This dynamic observation not only shed light on the behavior and interactions of HaTRPA1 in response to stimuli but also simplified the selection of the most suitable expression clone, enhancing the overall efficiency of the study.
Future studies should concentrate on refining the purification process and identifying more effective detergents to further deepen our comprehension of HaTRPA1 and explore its potential applications, including the development of pine weevil repellents to protect coniferous trees.

Popular Abstract

Have you ever wondered why do you feel a burning sensation while eating spicy food like horse radish or wasabi? The answer lies in a protein called Transient Receptor Potential Ankyrin 1 (TRPA1) that is activated by pungent natural products from mustard and allium plants. It plays an important role in the sensory nervous system, particularly in the detection of environmental irritants and noxious chemicals. It is found in many different types of cells throughout the body, including neurons, skin cells and immune cells.
Once TRPA1 is activated by irritants or other stimuli, a signal that is interpreted by our nervous system as pain or discomfort is sent. That response is designed to protect the body from further damage by causing a reflexive withdrawal or avoidance response. Interestingly, TRPA1 is also involved in other physiological processes beyond pain detection. For example, it has been implicated in regulating inflammation and immune function, as well as in the perception of certain tastes and smells.
Given its diverse function and widespread expression, TRPA1 is a promising target for the development of the new therapeutics to treat a range of conditions, from chronic pain to allergies and asthma. However, more research is needed to fully understand the complex mechanisms by which TRPA1 operates and to develop effective drugs that can selectively modulate its activity without causing unwanted side effects.
Hylobius abietis is a species of weevil belonging to the family Curculionidae, commonly known as the pine weevil. This insect can cause significant damage to trees, particularly conifers such as pine, spruce, and fir. The pine weevil’s harmful effects on trees can be attributed to several factors. One reason why H. abietis is considered bad for trees is its feeding behaviour. Adult pine weevils feed on the bark of young trees causing stunted growth and even death in severe cases. Additionally, the feeding activity of pine weevils can cause wounds to the tree’s bark, making it more vulnerable to disease and pest infestations.
It may be possible to fight the pine weevil by identifying a substance that can activate TRPA1, an agonist. When the pine weevil feeds on a plant, these chemical signals activate TRPA1 and potentially cause the insect to feel pain. By activating TRPA1, the insect’s feeding behaviour can be disrupted, potentially reducing damage to the plant.
In this study, the expression of TRPA1 linked to a green fluorescent protein (GFP) was performed successfully in yeast together with solubilization using Fos-choline-14. This detergent has been shown to effectively keep the protein stable in a solution, thus allowing for its purification using different kinds of chromatography. The process yielded a relatively pure protein from which the GFP was removed. To improve the stability of TRPA1, an attempt to exchange the detergent for an amphipol was made.
Further optimization of TRPA1 purification for structural studies may need to be considered, including the use of different detergents or amphipols.

• author: Kolodziejczyk, Oliwia
• supervisor: Urban Johanson
• organization: Department of Chemistry
• course: KEMR30 20231
• year: 2023
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• keywords: Biochemistry, membrane proteins, chromatography
• language: English
• id: 9124225
• date added to LUP: 2023-06-19 11:03:51
• date last changed: 2023-06-19 11:03:51