Nature Communications article by Prof. R. Manjunatha Kini: A general approach to protein folding using thermostable exoshells.

PProtein refolding is a complex procedure in bacterial recombinant expression. Aggregation and misfolding are the two challenges that can affect the overall yield and specific activity of the folded proteins. Thermostable exoshells (tES) are being considered as a novel nanoparticle solution for in vitro folding proteins. The tES are 12 nm diameter engineered particles derived from the thermophilic bacteria Archaeoglobus fulgidus with an eight nm aqueous cavity, a volume that can theoretically accommodate any molecules up to a volume of ~270 nm3. The shells, an assembly of 24 subunits contain 4.5 nm pores, which allow for easy permeation of solutes. A single mutation (Phe116 → His) at the three-fold symmetry axis disassembles them at pH 5.8 and assembles at pH 8.0. Twelve highly diverse proteins of interest (POIs) ranging from small toxins to human albumin, a dimeric protein (alkaline phosphatase), a trimeric ion channel (Omp2a), and the tetrameric tumor suppressor, p53 were folded successfully using tES. These proteins differed widely in their size, volume, disulfide linkages, isoelectric point, and multi versus monomeric nature of their functional units. Crude soluble yield (3-fold to >100-fold), functional yield (2-fold to >100-fold), and specific activity (3-fold to >100-fold) were increased for all the proteins tested. The average soluble yield was determined as 6.5 mg of folded protein per 100 mg of tES utilized. The electrostatic charge complementation between the tES interior and the protein substrate appears to be the primary determinant for functional folding.

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