New properties of inclusion bodies with implications for biotechnology.

Human G-CSF (granulocyte colony-stimulating factor) is a well-known biopharmaceutical drug being mostly produced by overexpression in Escherichia coli, where it appears in the form of IBs (inclusion bodies). Following our initial findings that properties of inclusion bodies strongly depend on the growth conditions used, especially growth temperature, we compared the characteristics of the G-CSF inclusion bodies prepared at two different temperatures, namely 42 and 25 degrees C. IBs formed at higher growth temperatures have properties similar to the usually described IBs, containing mainly denatured recombinant protein and being almost completely insoluble in aqueous solutions containing mild detergents or low concentrations of denaturants. In contrast, when produced at lower growth temperature of 25 degrees C, IBs show significantly different properties. Such IBs contain a significant proportion of G-CSF that is easily and directly extractable in the biologically active form, using non-denaturing solutions, which can be exploited for environmentally friendly biotechnological production. Irrespective of the production temperature, a significant decrease in IB volume was observed when transferring IBs from neutral to acidic (around 4) pH. Irreversible contraction of IBs at low pH was documented at the macro- and micro-scopic level using electron microscopy as a characterization tool. Together with volume decrease, a higher density, and thus decreased solubility, of IBs was observed at low pH, resulting in slower and less efficient extractability of the target protein.