Kloning, överuttryck och upprening av växtakvaporinet AtSIP1;1 i Escherichia coli

University essay from Lunds universitet/Examensarbeten i molekylärbiologi

Author: Johan Zhu; [2020]

Keywords: Biology and Life Sciences;

Abstract: Heterologous overexpression of proteins signifies that the encoding gene of a protein in one organism is expressed in another host organism that normally does not have that gene in its genome. In basic biochemical research, this is an important method for studying various protein functions such as activity and interactions in biological processes, as well as obtaining sufficient amounts to determine protein structures. However, the expression of a foreign gene may be inefficient due to a suboptimal match with the expression machinery, or suppressed as it consumes a lot of resources. It can also be incompatible due to deleterious effects on the host's cellular functions. In addition, it may be problematic to study certain types of proteins as these have properties that are difficult to handle. A class of proteins where research is very limited is membrane proteins, which include aquaporins (AQPs) that form water-permeable channels in various membranes of cells. These channels belong to the family Major intrinsic proteins (MIPs) and are particularly abundant in plants where they can be divided into three subfamilies, depending on their cellular location: Plasma membrane intrinsic proteins (PIPs), Tonoplast intrinsic proteins (TIPs), and NOD26-like intrinsic proteins (NIPs). However, a new subfamily of plant aquaporins has been discovered, which has been named Small basic intrinsic proteins (SIPs). The structure and function of SIPs have not been fully determined as it has proved difficult to overexpress members of this subfamily in previous research. The purpose of this project was to overexpress AtSIP1;1, which is one of three SIP isoforms present in the plant Arabidopsis thaliana, in the bacterial host Escherichia coli. This was achieved by subcloning the protein encoding gene sequence into an expression plasmid, optimize the expression conditions, purify the protein and examine the isolated protein. The outcome was that the protein could not be overexpressed in the host organism due to the use of defective materials in one of the methods, which caused problems to proceed with the project. Suggestions on updating the materials with newer ones or other types that can be used as reserves, as well as improving the work under sterile conditions have been made. For future studies, it has been proposed to include other host organisms with expression systems of varying complexity since an expression system with a simple composition in a lower organism may be very limited to be able to express a gene from a higher organism. Such expression systems involve those found in eukaryotic cells, which have a high degree of complexity and the ability to express proteins that are generally difficult to express.

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