Journal
PROTEIN SCIENCE
Volume 15, Issue 2, Pages 304-313Publisher
WILEY
DOI: 10.1110/ps.051813506
Keywords
high hydrostatic pressure; refolding; inclusion body; solutes; redox-shuffling agent; arginine
Categories
Ask authors/readers for more resources
High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three Gram-negative binding proteins (GNBP1, GNBP2, and GNBP3) from Drosophila, and two phosphatases from human were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in solubilization yields of similar to 42%-58% from 1 mg/mL of IBs. Addition of urea (I and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, and nonionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (similar to 70%-100%) to achieve refolding yields of similar to 55%-78% from IBs (I mg/mL). Our results suggest that the combination of HHP with arginine is most effective in enhancing the refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. Using the refolded proteins, the binding specificity of GNBP2 and GNBP3 was newly identified the same as with that of GNBP 1, and the enzymatic activities of the two phosphatases facilitates their further characterization.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available