Membrane Protein Expression, Purification and Characterization (mPEPC2)
Integral membrane proteins (IMPs) are encoded by 20-30 % of all open reading frames and provide key functions in the cell. Due to their hydrophobic nature, they are more difficult to study and structural information is lacking far behind their soluble counterparts. This course trains PhD students and early career postdocs with the latest developments in membrane protein production, characterization and structure determination methods. Modern protein expression techniques in various hosts will be presented and students will get acquainted with new crystallization, Cryo-EM and reconstitution techniques. An attitude to combine protein purification and characterization will be taught to impress the necessity on the students that proteins have to be conditioned with care to achieve structural information. The presence of a crystallization and cryo-EM facility as well as beamlines for SAXS and macromolecular crystallography at the venue will be employed to perform integrated structural biology experiments. The course has an emphasis on practicals in small groups, and a "Meet the scientist" session at the beginning of the course will ensure a close interaction between students and tutors. The aim is to inspire the students to apply the newly acquired knowledge and to maintain interactions among themselves and the tutors afterwards.
1. Expression of membrane proteins in HEK293/insect cells via transient transfection using GFP-fusion proteins.
2. Quality control of purified membrane protein preparations: nanoDSF and Blue Native PAGE.
3. Small Angle X-ray Scattering on integral membrane proteins.
4. Crystallization of integral membrane proteins in lipidic cubic phase.
5. Grid preparation for negative stain EM, data acquisition, analysis and low resolution 3D reconstruction.
6. Reconstitution of membrane proteins in proteoliposomes followed by functional analysis
7. Bioinformatics on membrane protein sequences and modelling of membrane protein structures.
8. Serial crystallography of small membrane protein crystals grown in Crystal Direct plates.
Accepted students are encouraged to bring their own samples (frozen state) for quality control, crystallization and negative stain EM. Precise shipment conditions and information will be provided to the selected applicants.
More Information: https://meetings.embo.org/event/22-mpepc2