Optimizing the Purification of the Painless Protein

Abstract

The mosquito Aedes aegypti is a known vector for many diseases, which leads to the use of harmful insecticides. Painless is a membrane protein that plays a role in sensing noxious heat mechanical stimuli, and by determining its structure, it could potentially be a target repellents as an alternative to insecticides.
By expressing Painless an additional eGFP HisTag in yeast cells, the protein can be extracted from the membrane and purified using metal affinity chromatography and size exclusion chromatography, while visualising the purification process through fluorescence. This would yield pure enough Painless to perform negative stain and cryogenic electron microscopy to determine its structure.
The results show that Painless easily degrades and aggregates, meaning a less than optimal yield of protein is obtained already in the first step of the purification. Continuous losses to aggregation and fragmentation over the purification process leads to complete loss of the intact protein. Results show that pH below 7 causes more aggregation, and indicates that pH around 9 may increase fragmentation. Increased detergent concentration does not yield higher Painless amounts either.
The results conclude that a broader investigation of Painless’ behaviour in different environments needs to be done, such as changing the salt concentration, glycerol contents or changing the detergent used, to find conditions where it is stable enough to allow purification.

Popular Abstract

Mosquitoes in the summer are not only annoying, but in many parts of the world actually pose a threat as they carry many diseases. To combat this, humans have used insecticides that, although efficient, causes harm to wildlife. It would therefore be great to have an insecticide that selectively repels the annoying mosquitoes, but without hurting wildlife.
This can be done through targeting the specific protein Painless, that sits on certain sensory cells of the mosquito. The Painless protein takes part in triggering the sensation of pain in the mosquito. By finding a target molecule that can stimulate the Painless protein, we can make the mosquito feel pain and be repelled without using harmful chemicals.
To find this stimulating molecule, we first need to visualised the protein and where a potential molecule can bind. We do this by forcing yeast cells to produce Painless with an added extension that also emits fluorescence and carries an infinity tag. Thanks to the affinity tag, we can use a set protocol using both the affinity of the tag, as well as size exclusion to purify the protein. However, it’s important to find the correct environment for the isolated protein to ensure it keeps its structure, which also is the aim of this lab.
The results show that the solubility of Painless likely is the reason to why the purification does not give high enough amounts of protein. We noticed clumping of the protein, fragmentation of the protein as well as persistent fluorescence on the affinity column. From that, its concluded that Painless does not prefer low pH environments, and instead prefers pH above 7. However, break down into smaller pieces is non-dependent on pH. Further studies should be done changing other factors, such as salt concentration, type of detergent or the use of glycerol to increase the stability of Painless.

• author: Häglund, Nora
• supervisor: Urban Johanson
• organization: Department of Chemistry
• course: KEML10 20251
• year: 2025
• type: M2 - Bachelor Degree
• subject: Chemistry
• keywords: biochemistry, Painless, membrane protein, purification
• language: English
• id: 9191648
• date added to LUP: 2025-06-05 13:21:34
• date last changed: 2025-06-05 13:21:34