Preparation & characterization of macromolecular complexes
This INSTRUCT/FRISBI practical course will provide participants with an overview of strategies for preparation and biophysical characterization of macromolecular complexes in view of biochemical and structural studies. The emphasis of the workshop will be placed on purification of macromolecular assemblies from endogenous sources using engineered cell lines and in reconstitution of multiprotein complexes using the baculovirus expression system.
The course is intended primarily for post‐doctoral researchers, PhD students and researchers in structural biology. Selected participants will improve their theoretical background and gain experience in designing experimental strategies relevant for their projects.
Hands on sessions
1/ Purification of complexes from engineered cell lines (Coordinator JP Concordet). Strategies to generate human knock‐in stable cell lines for isolation of difficult‐to‐express proteins and high resolution imaging. We will in particular give participants the opportunity to obtain a first hands‐on experience with CRISPR/Cas9 genome editing technology that will include design of genome editing experiment, selection of guide RNAs, donor DNA preparation, basic steps of KI cell line isolation and characterization.
2/ Production of recombinant multiprotein complexes using the baculovirus expression system (Coordinator A Poterszman). Construct and primer design, experimental setup and basic insect cell manipulations; Generation and amplification of recombinant baculoviruses; Expression screening and large scale production.
3/ Biophysical characterization and sample optimization (Coordinator C Birck) using complementary methods including nanoDSF, AUC‐SV, SEC‐MALS and EM analysis of negatively stained particles. Sample preparation, data acquisition and data analysis will be performed to characterize the homogeneity, size, shape, mass and stability of each sample.
Participants will be encouraged to present their projects, discuss bottlenecks, elaborate adapted strategies and will have the opportunity to characterize their own samples.
Mini-symposium - Wednesday Nov. 21st Afternoon
State‐of‐art strategies technologies for preparation and biophysical characterization of macromolecular complexes in view of biochemical and structural studies.
