Abstract
Preventing protein aggregation is a major goal of biotechnology. Since protein aggregates are mainly comprised of unfolded proteins, protecting against denaturation is likely to assist solubility in an aqueous medium. Contrary to this concept, we found denatured total cellular protein mixture from mammalian cell kept high solubility in pure water when the mixture was nucleic acids free. The lysates were prepared from total cellular protein pellet extracted by using guanidinium thiocyanate-phenol-chloroform mixture of TRIzol, denatured and reduced total protein mixtures remained soluble after extensive dialysis against pure water. The total cell protein lysates contained fully disordered proteins that readily formed large aggregates upon contact with nucleic acids or salts. These findings suggested that the highly flexible mixtures of disordered proteins, which have fully ionized side chains, are protected against aggregation. Interestingly, this unusual solubility is characteristic of protein mixtures from higher eukaryotes, whereas most prokaryotic protein mixtures were aggregated under identical conditions. This unusual solubility of unfolded protein mixtures could have implications for the study of intrinsically disordered proteins in a variety of cells.
Highlights
Proteins perform an extraordinary array of functions in cells [1]
Proteins are fractionated into the organic phase and precipitated by addition of 2-propanol (Fig. 1), and 90% of total cellular proteins can be recovered, which is considerably higher than was achieved by homogenizing cells directly in 8 M Urea (Fig. 2A)
After extensive washes with 0.3 M guanidine hydrochloride (GdnHCl)95% ethanol, or in ethanol, total cellular proteins were recovered as a tightly packed white pellet following centrifugation, which was used directly or stored at 220uC as a wet pellet to avoid the difficulties associated with resuspending dried pellets in denaturant solutions
Summary
Proteins perform an extraordinary array of functions in cells [1]. To understand the behavior of proteins in living cells, we must consider the extremely high intracellular concentrations of macromolecules. Some proteins exist in an intrinsically unstructured form, lacking stable secondary and tertiary structural elements, but retaining full functionality. IDPs lack bulky hydrophobic residues such as Ile, Leu, and Val, as well as aromatic residues such as Trp, Tyr, and Phe but are enriched in polar residues such as Arg, Gly, Gln, Ser, Pro, Glu and Lys, and the secondary structure-breaking amino acids Gly and Pro [6,7]. This composition results in high solubility in water despite being highly unstructured. The resultant soluble protein fractions are enriched in IDPs and are a valuable resource for proteomic research [13,14]
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