Experimental Approaches
We utilize a variety of biochemical approaches in an effort to unravel the molecular-level or atomic-level mechanisms that underlie protein function in health and disease. These approaches include:
- Recombinant protein expression in bacterial, insect and mammalian cell lines (small and medium scale)
- Protein purification and characterization (FPLC, SDS-PAGE etc)
- Gene cloning and expression vector construction
- Biophysical and structural analysis of proteins
- Enzymology and assay development
- Proteomics and peptide sequencing
- Specialized cell-based assays
Some examples can be found below:
SDS-PAGE showing the expression of the aminopeptidase ERAP1 in the medium of growing Hi5 cells and its purification by affinity chromatography:
Protein stability against chemical denaturation followed by tryptophan fluorescence:
Circular Dichroism Spectroscopy for the investigation of the secondary structure of studied proteins
X-ray crystallography is a powerful tool for the investigation of enzymatic mechanisms. Starting with large amounts of pure protein one needs to first obtain protein crystals (usually very small, in the order of 0.1-0.5 mm) through a screening process that allows the identification of the optimal crystallization conditions. An example of protein crystals is shown to the left.
Exposing these crystals to x-ray radiation, produces a diffraction pattern that contains information on the arrangement of the protein atoms in three-dimensional space
Mathematical processing of these data (Fourier Transform) eventually yields a 3D map of the electron density of the molecule (below in yellow). Building the known primary structure of the protein into this electron density map, yields a 3D model of the protein structure (red). Knowing the 3D structure of the protein greatly helps understand how it works.
A custom fluorigenic assay for the detection of aminopeptidase activity on large peptides is shown here. In this assay the aminopeptidase excises an internally quenched tryptophan residue from the peptide leading to an enhancement in solution fluorescence that can be quantified. The rate of solution fluorescence enhancement corresponds to the enzyme activity.
Fluorescence-activated cell sorting can be used to separate appropriately labeled live cells and quantitate cell-surface markers of antigen presentation:
