Overexpression and purification of TRPA1 from Anopheles gambiae with GFP-tag
(2023) KEML10 20231Department of Chemistry
- Abstract
- Introduction: The topic of this project was the expression and purification of Transient Receptor Potential Ankyrin 1 (TRPA1) from Anopheles gambiae, both the full length protein and a truncated version.
Background: The structure of AgTRPA1 has not previously been determined, a step towards the determination is to express and purify the protein. Particular challenges working with AgTRPA1 are that it is a membrane protein which is notorious for being expressed at a low level and the need of detergent to get the protein solubilized.
Aim: The aim with this project was to transform a plasmid containing the protein sequence into the yeast Pichia pastoris where it would be expressed. The protein was then purified using a his-tag and followed... (More) - Introduction: The topic of this project was the expression and purification of Transient Receptor Potential Ankyrin 1 (TRPA1) from Anopheles gambiae, both the full length protein and a truncated version.
Background: The structure of AgTRPA1 has not previously been determined, a step towards the determination is to express and purify the protein. Particular challenges working with AgTRPA1 are that it is a membrane protein which is notorious for being expressed at a low level and the need of detergent to get the protein solubilized.
Aim: The aim with this project was to transform a plasmid containing the protein sequence into the yeast Pichia pastoris where it would be expressed. The protein was then purified using a his-tag and followed with a GFP-tag.
Methods: Electroporation was used to transform the yeast, the expression of protein was screened with the fluorescence of GFP. A jackpot clone was grown in a fermenter and cells were lysed with a bead beater, the protein was solubilized with foscholine-14 and purified with Immobilized metal affinity chromatography, reverse IMAC and size exclusion chromatography.
Results: The truncated version of AgTRPA1 was successfully expressed and purified with the method mentioned above. Expression of the full length protein could not be confirmed due to lack of time and therefore the purification method could not be tested on this version. Purification with the his-tag was an effective method and the GFP-tag made it possible to follow the protein through the entire process.
Conclusion: From this project, it was concluded that the truncated version of the protein was easier to transform into the cell compared to the full length. Only the expression of the truncated protein could be confirmed. The purification of the truncated protein was successful and a high amount was purified. (Less) - Popular Abstract
- TRPA1 from malaria mosquito
In the membrane of the cell there are proteins embedded that works as channels between the outside and inside of the cell. These proteins are used to communicate the environment outside of the cell to the inside of the cell. One protein like this is the so called wasabi receptor which has the more technical name Transient Receptor Potential ion channel with ankyrin repeats (TRPA1). This type of protein can be found in a number of organisms, but in this project the focus is on TRPA1 from the African malaria mosquito. There are substances, e.g. in wasabi, which can bind to this protein and activate a pain response. To design a substance that generates the pain response specifically in the mosquito, the protein... (More) - TRPA1 from malaria mosquito
In the membrane of the cell there are proteins embedded that works as channels between the outside and inside of the cell. These proteins are used to communicate the environment outside of the cell to the inside of the cell. One protein like this is the so called wasabi receptor which has the more technical name Transient Receptor Potential ion channel with ankyrin repeats (TRPA1). This type of protein can be found in a number of organisms, but in this project the focus is on TRPA1 from the African malaria mosquito. There are substances, e.g. in wasabi, which can bind to this protein and activate a pain response. To design a substance that generates the pain response specifically in the mosquito, the protein structure needs to be known. This means that with knowledge of this protein a repellent for the mosquito can be designed.
For this project, the goal was to achieve a pure protein sample. To study this protein, it first had to be produced, which was done by introducing the DNA sequence for the protein into a fast growing organism such as a yeast. To visualise the protein throughout the process, the sequence for a green fluorescent protein was added to the protein sequence so that the protein would emit green light when illuminated with blue light. This fluorescent light was used to evaluate which yeast colonies that produces the highest amount of the protein. A colony was chosen to grow in big scale and the cells were broken and the TRPA1 was purified from the other parts of the cells.
The results from this project were that the full length protein was hard to introduce into the yeast and it was produced at a low level. However, a shortened version of the protein was successfully introduced and produced in large quantities, therefore the purification was done on this version. To conclude this project, the method chosen worked for the shorter version of the protein, but more time is needed in order to determine if the same method can be used for the full length protein. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9118991
- author
- Edström, Elin LU
- supervisor
- organization
- course
- KEML10 20231
- year
- 2023
- type
- M2 - Bachelor Degree
- subject
- keywords
- AgTRPA1, Biochemistry, GFP-tag, His-tag, Pichia pastoris
- language
- English
- id
- 9118991
- date added to LUP
- 2023-06-08 11:28:00
- date last changed
- 2023-06-08 11:28:00
@misc{9118991,
abstract = {{Introduction: The topic of this project was the expression and purification of Transient Receptor Potential Ankyrin 1 (TRPA1) from Anopheles gambiae, both the full length protein and a truncated version.
Background: The structure of AgTRPA1 has not previously been determined, a step towards the determination is to express and purify the protein. Particular challenges working with AgTRPA1 are that it is a membrane protein which is notorious for being expressed at a low level and the need of detergent to get the protein solubilized.
Aim: The aim with this project was to transform a plasmid containing the protein sequence into the yeast Pichia pastoris where it would be expressed. The protein was then purified using a his-tag and followed with a GFP-tag.
Methods: Electroporation was used to transform the yeast, the expression of protein was screened with the fluorescence of GFP. A jackpot clone was grown in a fermenter and cells were lysed with a bead beater, the protein was solubilized with foscholine-14 and purified with Immobilized metal affinity chromatography, reverse IMAC and size exclusion chromatography.
Results: The truncated version of AgTRPA1 was successfully expressed and purified with the method mentioned above. Expression of the full length protein could not be confirmed due to lack of time and therefore the purification method could not be tested on this version. Purification with the his-tag was an effective method and the GFP-tag made it possible to follow the protein through the entire process.
Conclusion: From this project, it was concluded that the truncated version of the protein was easier to transform into the cell compared to the full length. Only the expression of the truncated protein could be confirmed. The purification of the truncated protein was successful and a high amount was purified.}},
author = {{Edström, Elin}},
language = {{eng}},
note = {{Student Paper}},
title = {{Overexpression and purification of TRPA1 from Anopheles gambiae with GFP-tag}},
year = {{2023}},
}