ABOUT THE PRACTICAL COURSE
This intensive EMBO Practical Course is dedicated to modern methods in sample preparation and characterization of proteins for structural studies. It covers cloning and overexpression in bacterial and eukaryotic systems, protein purification with and without tags, sample quality control, sample optimization and crystallization.
This is a hands-on course for researchers in the early stages of their career who wish to learn molecular biology techniques in sample preparation and characterization for structural studies. It is centered around parallel practicals in small groups with experts using model systems. Participants are also encouraged to bring their own samples for protein expression, optimization and crystallization.
The overexpression of proteins needs to be carefully designed to obtain a significant amount of properly folded material. In this module different protein expression strategies are presented including the following practicals:
- Cloning and choice of expression vectors: A 3 hour computer tutorial on the in-silico design of expression constructs using several software tools
- Bacterial expression: A 2x3 hour tutorial on the screening of constructs using different solubility and purification tags in E. coli
- Baculovirus expression in insect cells: A 2x3 hour practical on the preparation of baculovirus titer and small scale expression in insect cells. Participants are offered the possibility to have their gene of interest cloned into the pOPIN vector system. When the cloning has proven successful, the protein will then be expressed in E. coli and possibly insect cells prior to the course. Results will be discussed during the course. Please indicate on your application if you are interested in this option.
The purification of proteins with and without an affinity tag require different techniques, and we have two practicals that cover two difficult scenarios:
- Expression and purification of membrane proteins in E. coli A 2x3 hour practical on protein purification and crystallisation of a proton pump
- Purification of tag-less proteins using a range of separation techniques. A 2x3 hour practical that covers traditional purification techniques
The biophysical characterization of purified protein can provide a quality assessment of the sample and it can offer complementary knowledge on the system. The main topic of this module is crystallization of macromolecules, and especially how to optimize marginal crystallization conditions to obtain diffracting crystals. There are the following practicals:
- A 3 hour practical on Thermofluor-based thermal stability measurements using optimization screens
- A 3 hour practical on Quality control of samples using mass spectrometry and light scattering
- A 3 hour practical in crystal scoring and optimization
- A 3 hour practical on vapour diffusion based crystallization
Participants are also offered the possibility to bring their purified samples for crystallization to the high-throughput crystallization facility. A more extensive characterization of samples is sponsored throughout 2014 through Biostruct-X.
EMBL c/o DESY
We look forward to welcoming you