Laboratory scale production of complex protein substrates using substrate-complimentary nanoenvironments

Abstract In vitro protein folding is a complex process which often results in protein aggregation, low yields and low specific activity. We report the use of nanoscale exoshells (tES) to provide specific nanoenvironments for the folding and release of 12 highly diverse protein substrates ranging from small protein toxins to human albumin, a dimeric protein (alkaline phosphatase), a trimeric ion channel (Omp2a) and the tetrameric tumor suppressor, p53. These proteins represent a unique diversity in 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 of POI was 6.5 mg/100 mg of tES. Charge complementation between the tES internal cavity and the protein substrate was the primary determinant in functional folding. Our results confirm the importance of nanoscale electrostatic effects and provide a novel nanoparticle solution for folding proteins in vitro.