Marjan Bele

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 °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 °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.

Zinc bioaccumulation in a terrestrial invertebrate fed a diet treated with particulate ZnO or ZnCl2 solution.

A number of reports on potential toxicity of nanoparticles are available, but there is still a lack of knowledge concerning bioaccumulation. The aim of this work was to investigate how different sources of zinc, such as uncoated and unmodified ZnO nanoparticles, ZnCl(2) in solution, and macropowder ZnO influence the bioaccumulation of this metal in the terrestrial isopod Porcellio scaber. After exposure to different sources of Zn in the diet, the amount of assimilated Zn in whole body, the efficiency of zinc assimilation, and bioaccumulation factors (BAFs) were assessed. The bioaccumulation potential of Zn was found to be the same regardless of Zn source. The amount of assimilated Zn and BAF were dose-dependent, and Zn assimilation efficiency was independent of exposure concentrations. The Zn assimilation capacity was found to be up to 16% of ingested Zn. It is known that as much as approximately 20% of Zn can be accreted from ZnO particles by dissolution. We conclude that bioaccumulation of Zn in isopods exposed to particulate ZnO depends most probably on Zn dissolution from ZnO particles and not on bioaccumulation of particulate ZnO.

Suspensions of modified TiO2 nanoparticles with supreme UV filtering ability.

TiO2nanoparticles (20–23 nm in size) were coated with a silica layer onto which lauric acid was strongly bound. The strong (probably covalent) bonding was proved using detailed comparative thermal analysis. The functionalized TiO2nanoparticles exhibited significantly reduced agglomeration, both in dry and in dispersed states (in oily media). The reduced tendency towards agglomeration was consistent with contact angle measurements which showed an increased hydrophobicity of functionalized TiO2. The strong (covalent) bonding also greatly improved the stability of the nanoparticulate sample in suspensions. Finally, the UV filtering efficiency of functionalized samples was much improved when compared to non-functionalized or those functionalized using conventional stabilizers based on adsorption. This effect was consistent with our theoretical prediction in which we correlated the particle size and the UV filtering ability.

Nonclassical inclusion bodies in Escherichia coli

Background Formation of inclusion bodies (IBs) during over-expression of recombinant proteins in Escherichia coli is of common occurrence. The target protein inside inclusion bodies is usually misfolded and a series of denaturation/ renaturation steps is necessary for isolation of biologically active protein. Purification protocol must be optimized case by case. Due to low efficiency of most denaturation/ renaturation procedures a lot of trials have been performed to obtain soluble and properly folded target proteins inside E. coli cytoplasm with the aim of increasing the yield of the target protein.