FRENCH INFRASTRUCTURE FOR INTEGRATED STRUCTURAL BIOLOGY
The first for macromolecule purification after over-expression, the macromolecule has to be extracted from the bacteria containing it. Several methods of cell lysis are commonly used to achieve this : enzymatic disruption (lysozyme), sonication using high frequency sound waves, liquid homogenization and freeze/thaw cycles.
The choice of the lysis method depends on the type of cells (E. coli only at the CBS), the volume of cell suspension and the fragility of the proteins to be recovered. The composition of the lysis buffer is also crucial to maintain the proteins of interest in the soluble fraction after cell breakage.
Protein purification consists of a series of separation steps intended to isolate a single type of protein from a complex mixture. Separation steps may exploit differences in protein size, physicochemical properties, binding affinity and biological activity. It usually take advantage of an affinity purification step thanks to the expression of affinity-tagged proteins (His-tag, MBP-, GST- or thioredoxin-fusions, ...).
High-performance purification is performed on preparative or analytical scale columns using chromatographic systems. Usually the proteins are detected as they are eluted from the column by their absorbance signal.
Preparative purifications aim to produce a relatively large quantity of purified proteins (> 10 mg) for subsequent structural and functional studies. Analytical purifications produce a relatively small amount of proteins (0.1 - 1 mg) for a variety of research purposes and are commonly used to set up a purification protocol.
Different chromatographic systems are available for large and analytical purification of proteins and macromolecular complexes using a variety of prepacked columns and chromatography media.