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Exploring the activity of tobacco etch virus protease in detergents

Mezher, Amar (2017) MOBK01 20162
Degree Projects in Molecular Biology
Popular Abstract
Exploring the potential of biotechnological Tools

Protein production has always been a vital part in many studies, which has enabled us to produce proteins that are e.g. used for medicinal purposes, such as insulin. In order to achieve this, one must first obtain an adequate amount of protein. The production of proteins is achieved by utilizing bacteria, which can produce foreign proteins from introduced genes. Once the bacteria have produced the protein of interest, how does one isolate it from the rest of the proteins in the bacteria cell?

When the protein of interest is expressed in the bacteria, it is produced with an attached part that is called an affinity tag. These affinity tags are used to isolate the protein from the rest... (More)
Exploring the potential of biotechnological Tools

Protein production has always been a vital part in many studies, which has enabled us to produce proteins that are e.g. used for medicinal purposes, such as insulin. In order to achieve this, one must first obtain an adequate amount of protein. The production of proteins is achieved by utilizing bacteria, which can produce foreign proteins from introduced genes. Once the bacteria have produced the protein of interest, how does one isolate it from the rest of the proteins in the bacteria cell?

When the protein of interest is expressed in the bacteria, it is produced with an attached part that is called an affinity tag. These affinity tags are used to isolate the protein from the rest of impurities that are inside the bacterial cell. The way affinity tags work can be explained with the key and lock model. A particular column (Figure 1), which contains a material that binds the affinity tag, is used when purifying the protein of interest. When the mixture of proteins from the opened bacteria cell is added to the column, only the correct key, the affinity tag with the protein, will fit into the lock. The other components in the mixture will wash away, which leaves the protein of interest isolated from the rest. However, even though affinity tags are of great use, they have been known to affect the biological function of the protein they are attached to. Therefore they are sometimes removed after they have served their purpose. For this reason, proteases are used, which act as scissors and cut off affinity tags.

Tobacco Etch Virus Protease
One of the sharpest proteases that is used today, is the tobacco etch virus (TEV) protease. TEV has generally been used for cutting off affinity tags, and works well with most proteins, but not all proteins. Some proteins cannot be solubilized in water, which is required so that TEV can cut off their affinity tag. For this, detergents are used, which solubilizes the proteins, but it has been reported that some detergents inhibit TEVs function. It is therefore crucial to explore if there are any suitable detergents that can be used with TEV, so that its potential is fully utilized. The aim of our work was to examine this, and see if we could find any detergents that would retain TEVs function and allow it to be used with a new set of proteins.
The result for the study of TEVs activity in five different detergents can be seen in Figure 2. In this analysis, a gel which separates larger proteins from smaller, from top to bottom was used. It shows which detergents were successful with TEV in order to cut a protein into two smaller pieces, which can be seen as two stacking blue bands in each lane of lane 1-3. Here we can see that we have found three detergents that have been successful, and two detergents (lane 4-5) that disrupted TEVs activity. We now know which detergents to use with TEV for future studies.

Supervisor: Urban Johansson
Degree Project, 30 credits, in Molecular Biology, 2016
Department of Biology/ Biochemistry and Structural Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Mezher, Amar
supervisor
organization
course
MOBK01 20162
year
type
M2 - Bachelor Degree
subject
language
English
id
8901710
date added to LUP
2017-01-30 15:43:40
date last changed
2017-01-30 15:43:40
@misc{8901710,
  author       = {Mezher, Amar},
  language     = {eng},
  note         = {Student Paper},
  title        = {Exploring the activity of tobacco etch virus protease in detergents},
  year         = {2017},
}