Refolding of endostatin from inclusion bodies using high hydrostatic pressure.
High hydrostatic pressure was used for concomitant solubilization and refolding of insoluble endostatin (ES) aggregated as inclusion bodies (IBs). High hydrostatic pressure (200 MPa or 2 kbar) was applied in combination with nondenaturing concentrations of guanidine hydrochloride. High levels of correctly folded ES (90 mg/L culture) were obtained after optimization/standardization of the procedure by applying pressures of 200 MPa for 16 h in 1.5 M guanidine hydrochloride/0.5 mM oxidized glutathione and reduced glutathione. Refolded ES was purified by affinity chromatography on a heparin column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, size exclusion HPLC, circular dichroism, and intrinsic fluorescence. We demonstrated that high pressure can successfully convert insoluble IBs of ES expressed in Escherichia coli into an ES preparation with native tertiary structure and full biological activity.
Chura-Chambi
Rosa Maria
RM
Instituto de Pesquisas Energéticas e Nucleares, Instituto de Pesquisas Energéticas e Nucleares-CNEN/SP, São Paulo, Brazil.
Genova
Luis Antonio
LA
Affonso
Regina
R
Morganti
Ligia
L
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006874:Hydrostatic Pressure; D002479:Inclusion Bodies / Q000378:metabolism*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D043169:Endostatins