Overexpression and Purification of Mouse Divalent Metal Transporter 1 (mDMT1) from Saccharomyces cerevisiae

Abstract

The membrane integrated divalent metal transporter 1 (DMT1) functions as a transporter of mainly iron across cellular membranes within the human body. Studies indicate that dysregulated transport by DMT1 causes accumulation of iron within the midbrain leading to neuronal cell death – a common symptom among patients suffering from Parkinson’s disease (PD). Detailed structural information of DMT1 is currently lacking but may aid in drug development for PD. To solve the 3D crystal structure of DMT1, high-purity mono disperse protein must first be obtained. To this end, we expressed mouse DMT1 (mDMT1) in Saccharomyces cerevisiae and developed protocols to purify this protein. Based on a detergent screen and our purification trials we find that mDMT1 is soluble in n-dodecyl-β-D-maltpyranoside (DDM) and an addition of cholesteryl hemisuccinate (CHS) increases solubility of the protein in solution. Furthermore, we determine that the highest protein concentration by S. cerevisiae is obtained at 23 °C after 43 h. Although none of the performed purification trials was entirely successful at yielding mDMT1 samples suitable for crystallization, we present guidelines as to what areas of the protocol may be optimized to achieve this goal.

Popular Abstract

Crystals – A Possible Cure for Parkinson’s Disease?

In labs all over the world, scientists are trying to understand why some of us suffer from different kinds of diseases. Perhaps more importantly, scientists are also trying to find a cure for them. Experiments, performed at Lund’s University, aim to generate crystals from of a protein called divalent metal transporter 1 (DMT1) that is suspected to cause Parkinson’s disease. These crystals may possess information about DMT1 that paves the way for novel drugs curing Parkinson’s patients.

It is hard to imagine how a crystal, built by proteins, could be of help to produce drugs and cure diseases. Instead, imagine the pathway from wheat in a field to bread in your kitchen. First, you would have to grow a lot of wheat. Secondly, you would have to harvest it all and grind it into a powder. Before making bread out of the powder, it would be necessary to purify it from the husk and other contaminants such as soil and insects, in order to obtain a pure batch of flour. Likewise, this is the procedure when creating a crystal as the final product. Crystals must be made from a pure batch of proteins that were produced within many thousands of cells. You may think of the cells as wheat, something that primarily has to be grown and secondly harvested. The main goal for creating crystals however, is not to fill an empty stomach, but to reveal the 3D-structure from the protein that it maintains. Furthermore, this 3D-structure may be of huge advantage in order to develop cures against a wide range of diseases.

A clean way to crystal richness
Now, let us narrow down a bit and focus on the steps in-between cell harvesting and the pure sample of protein. In the same way that it is pointless to bake bread with dirty flour, it is the same to create crystals with impure protein. Unfortunately, there is no golden recipe for how to obtain pure protein. In fact, it may take years to live up to crystal friendly criteria. So, what methods were tried at Lund’s university in order to reach them?

Complementary properties, such as something that is called a His-tag, was added to the DMT1 by gene modifying techniques. His-tags give proteins one great property – they start to love metals like nickel more than any other protein does. A second feature added was nothing less than fluorescence! These features make it is possible to separate the nickel attracted protein from unwanted ones and to keep track of it throughout the purification process by fluorescence detection.

Generally speaking, proteins are built up in a complex structure that is sensitive to extraneous changes. Hence, many purification setups were performed with varying parameters, such as pH, temperature and buffer composition, in order to find a condition where the protein thrive. This far, with success in finding a condition where the cells produce a lot of the protein and a buffer composition where it is kept stable. Numerous steps are left before crystal friendly DMT1 samples are obtained. But do not worry! Many more methods remains to try and thus the lab-work aiming to defeat Parkinson’s disease continues.

Advisor: Susanna Törnroth Horsefield
Degree Project in Molecular Biology, 30 credits 2016
Department of Biology, Lund University

• author: Arbab, Olivia
• supervisor: Susanna Horsefield
• organization: Degree Projects in Molecular Biology
• course: MOBY01 20161
• year: 2016
• type: M2 - Bachelor Degree
• subject: Biology and Life Sciences
• language: English
• id: 8892046
• date added to LUP: 2016-09-19 14:51:38
• date last changed: 2016-09-19 14:51:38