Figure 2. General strategy used for production of stable complexes.

(a) Flowchart describing the 9 steps used in the pipeline. After bioinformatics analysis (a1) to define protein domains (bI), a DNA library is created. The proteins are then cloned in fusion with solubility and affinity tags (bII). Next they are transferred into expression vectors (a3) for protein production in prokaryotic and eukaryotic cells (bIII). Two strategies are then tested. One is the production of each protein individually (a4,bIV) and complex reconstitution by dialysis (a, line Ia) or co-cell lysis (a, line Ib) and another is the co-expression of the different proteins together (a, line II; bV). After solubility tests, steps 3–5 are recycled until the optimal conditions for the proteins/complex solubility and stability are found. After a large scale production (a6) protein purification (a7, line Ia) and complex reconstitution (a8, line Ia) the complex is purified by affinity purification and size exclusion chromatography.