New properties of inclusion bodies with implications for biotechnology

Abstract

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.

A Structural Study of CESA1 Catalytic Domain of Arabidopsis Cellulose Synthesis Complex: Evidence for CESA Trimers

“…Attempts to solubilize AtCESA1CatD under denaturing conditions using urea or lithium dodecyl sulfate were unsuccessful, resulting in protein aggregation during the refolding process. However, the protein was solubilized under non-denaturing conditions using a low concentration of sodium lauroyl sarcosine according to a previously reported protocol (García-Fruitós et al, 2005;Jevsevar et al, 2005;Peternel et al, 2008). The purified protein was .95% pure after size exclusion chromatography as judged by SDS-PAGE analysis (Fig.…”

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

“…It has been demonstrated that inside an IB there are heterologous and host proteins combining native-like structures with partially folded and misfolded proteins [13,[16][17][18][19][20][21]. The IB formation and its maintenance involve a complex network of intracellular responses related to culture conditions, leading to complex and stable structures sometimes showing bioactivity [13,22].…”

“…The IB formation and its maintenance involve a complex network of intracellular responses related to culture conditions, leading to complex and stable structures sometimes showing bioactivity [13,22]. Due to their different physicochemical properties, IBs have been proposed for various uses, such as catalysts, support materials, drug delivery agents, cell therapy, and immunogens, and their recent application has become an important new topic in biology, medicine and biotechnology [11,[23][24][25][26][27][28][29][30].…”

“…IBs are dynamic reservoirs that contain a large amount of recombinant protein, various host proteins like chaperones, among other components of the cytoplasm [4,8]. IBs are highly hydrated dense particles of porous structure [9,10], their surface varies from rough to smooth [6], and their size is normally in the range of 50 to 700 nm, having spherical, cylindrical or ellipsoidal teardrop shapes [10][11][12][13][14][15].…”

Engineering protein self-assembling in protein-based nanomedicines for drug delivery and gene therapy

“…Additionally, these protein-based nanoparticles are mechanically stable (García-Fruitós et al, 2009), showing regulatable size (García-Fruitós et al, 2009), geometry (Garcia-Fruitos, Seras-Franzoso, Vazquez, & Villaverde, 2010, density (Peternel, Jevsevar, Bele, Gaberc-Porekar, & Menart, 2008), Z-potential, stiffness and wettability (Diez-Gil et al, 2010). Importantly, it is feasible to obtain IBs formed by almost any protein of interest, eventually assisted by pull-down peptides that act as fuzzy architectonic tags Garcia-Fruitos et al, 2005).…”