Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure.
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 approximately 42%-58% from 1 mg/mL of IBs. Addition of urea (1 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 (approximately 70%-100%) to achieve refolding yields of approximately 55%-78% from IBs (1 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 GNBP1, and the enzymatic activities of the two phosphatases facilitates their further characterization.
Lee
Seung-Hyun
SH
Department of Molecular Science and Technology, Ajou University, San 5, Woncheon-dong, Yeongtong-gu, Suwon, Korea.
Carpenter
John F
JF
Chang
Byeong S
BS
Randolph
Theodore W
TW
Kim
Yong-Sung
YS
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001120:Arginine / Q000494:pharmacology*; D002352:Carrier Proteins; D004229:Dithiothreitol / Q000378:metabolism*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000378:metabolism; D054637:Dual-Specificity Phosphatases; D019803:Glutathione Disulfide / Q000378:metabolism*; D006801:Humans; D006874:Hydrostatic Pressure; D002479:Inclusion Bodies / Q000737:chemistry* / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases; D011487:Protein Conformation; D017510:Protein Folding; D020673:Protein Renaturation; D017027:Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility
D000817:Anilino Naphthalenesulfonates; D002352:Carrier Proteins; D029721:Drosophila Proteins; C480085:Gnbp1 protein, Drosophila; D011994:Recombinant Proteins; D001120:Arginine; D010749:Phosphoprotein Phosphatases; C107523:DUSP7 protein, human; D054637:Dual-Specificity Phosphatases; C516883:PTPRS protein, human; D017027:Protein Tyrosine Phosphatases; D054623:Receptor-Like Protein Tyrosine Phosphatases, Class 2; D004229:Dithiothreitol; D019803:Glutathione Disulfide