Resumen de Expression and characterization of a human sodium glucose transporter (hSGLT1) in Pichia pastoris /
In the last 20 years, the characterization of membrane protein has become an interesting field for drug design but, the lack of information regarding their mechanism of action makes it even harder to screen for new drug targets. Therefore, studying the structure and mechanism of action of membrane proteins should accelerate the research of new drugs and its medical implications.
The project presented here is focused on exploring the features of a human sodium glucose co-transporter (hSGLT1). This transport is involved in absorbing and reabsorbing the glucose in the intestine and it’s linked and directly involved in some diseases like: glucose galactose malabsorbation (GGM), diabetes and even some mental diseases like Alzheimer’s diseases. The mechanism of action of this transporter is partially understood although some features are still under debate and unclear. However, the 3D structure of this transporter is unknown and it’s an end goal for elucidating the mechanism of action of the transporter which will allow screening easily more effective drugs.
In order to be available to crystalize a membrane protein for its structure, a huge number of initial steps are necessary. First, the protein of interest must be expressed in a recombinant system satisfactorily and, membrane proteins are not easily expressed in all expression systems. Second, human membrane proteins are even harder to express because it’s necessary to work with a non-prokaryote expression system or, otherwise, it is very likely to obtaining in the end a non-functional protein. It has been described, only once, that hSGLT1 could be expressed in a eukaryotic yeast expression system: Pichia pastoris. This yeast has been used a lot in the last 10 years for expressing membrane proteins successfully because, unlike Saccharomyces cerevisae, it can grow much more and therefore obtain more protein in the end. Another advantage is the stability of the expression clones of P. pastoris which eventually leads to a more reproducible result. Although, some disadvantages must be overcome at the same time to have good expression levels like, for example, the clone selection. Because of all of this, the project presented here was initially based on this previous work done in P. pastoris.
This thesis is focused on the expression, purification and characterization of hSGLT1 expressed in a yeast heterologous expression system (P.pastoris). Initially, two vectors were design: One expressing hSGLT1 fused to eGFP and another construct without eGFP. The eGFP construct was used to monitorize the protein expression faster and easier which allows optimizing the protein expression by screening for best expression conditions like: induction time, temperature, media and more. Membrane proteins required to be extracted from the membrane in order to purify them and, to do so; a detergent is required. The eGFP fused protein was also used to screen which detergent is more suitable for protein extraction too. Although, both expression product (non-GFP and GFP) were purified and isolated only the WT hSGLT1 was used for further analysis. Despite the difficulties to reproduce what was described before, the protein was eventually subjected to a binding assay.
The binding assay was done with an unusual technique which is: voltage-clamp in planar lipid membranes. This technique allowed to measure transport (functionality) of the protein without the need to be incorporated in a liposomes.
The main conclusion extracted in this work is that a functional hSGLT1 can be purified which allows a wide number of possibilities in the future like crystallization for elucidating the structure and mechanism of the protein.
