Schematic workflow of the protein purification protocol development. Biomass for the intracellular production strains was extracted by various PB harvesting techniques and eventually solubilised under many different conditions requiring stepwise optimisation. The effects at each step were measured by Western blotting for yield increase and by activity assay, to determine whether integrity of the GLA enzyme fusion was retained. The harvesting, PB treatment and solubilisation methods tested are shown in the arrows, and the final solubilisation conditions are shown in the circle on the top. A plus sign (+) represents an increase in active yield and two or three such signs show the approximate magnitude of this effect. Similarly one to three negative signs (-) denote the magnitude of active yield decrease. A null effect sign (Ø) was used where there was no apparent change in active yield. Some additives have both signs to show that the effect of active yield was dictated by a change in molarity of the additive.