Significance: Hydrophobicity governs a wide range of fundamental physicochemical processes, but its physical origin is unclear. The classical explanation of hydrophobicity is that tiny “icebergs” are formed near such solutes; however, no experimental proof has been advanced for their existence. Here, we used four small purely hydrophobic solutes (methane, ethane, krypton, and xenon) in water to study hydrophobicity at the most fundamental level. We present unequivocal experimental proof for strengthened water hydrogen bonds near purely hydrophobic solutes, matching those in ice and clathrates. The water molecules involved in the enhanced hydrogen bonds display extensive structural ordering resembling that in clathrates, thus indicating the fundamental interconnection between electrostatic screening (shielding) and the hydrophobic effect.

Human granulocyte-colony stimulating factor (hG-CSF), an important biopharmaceutical drug used in oncology, is currently produced mainly in Escherichia coli. Expression of human hG-CSF gene in E. coli is very low, and therefore a semisynthetic, codon-optimized hG-CSF gene was designed and subcloned into pET expression plasmids. This led to a yield of over 50% of the total cellular proteins. We designed a new approach to biosynthesis at low temperature, enabling the formation of "nonclassical" inclusion bodies from which correctly folded protein can be readily extracted by nondenaturing solvents, such as mild detergents or low concentrations of polar solvents such as DMSO and nondetergent sulfobetaines. FT-IR analysis confirmed different nature of inclusion bodies with respect to the growth temperature and indicated presence of high amounts of very likely correctly folded reduced hG-CSF in nonclassical inclusion bodies. The yield of correctly folded, functional hG-CSF obtained in this way exceeded 40% of the total hG-CSF produced in the cells and is almost completely extractable under nondenaturing conditions. The absence of the need to include a denaturation/renaturation step in the purification process allows the development of more efficient processes characterized by higher yields and lower costs and involving environment-friendly technologies. The technology presented works successfully at the 50-L scale, producing nonclassical inclusion bodies of the same quality. The approach developed for the production of hG-CSF could be extended to other proteins; thus, a broader potential for industrial exploitation is envisaged.

For a long time IBs were considered to be inactive deposits of accumulated target proteins. In our previous studies, we discovered IBs containing a high percentage of correctly folded protein that can be extracted under non-denaturing conditions in biologically active form without applying any renaturation steps. In order to widen the concept of correctly folded protein inside IBs, G-CSF (granulocyte colony stimulating factor) and three additional proteins were chosen for this study: GFP (Green fluorescent protein), His7dN6TNF-α (Truncated form of Tumor necrosis factor α with an N-terminal histidine tag) and dN19 LT-α (Truncated form of Lymphotoxin α).