Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography.
A procedure for renaturation of heterodimeric platelet-derived growth factor (PDGF-AB) from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography is described. Either prepurified or crude PDGF-AB inclusion bodies solubilized with guanidinium hydrochloride were subjected to buffer exchange from denaturing to renaturing conditions during chromatography. Renaturation of PDGF-AB involves folding of the solubilized and unfolded molecules into dimerization competent monomers during size-exclusion chromatography and subsequent dimerization of folded monomers into the biologically active heterodimeric growth factor. Optimized conditions result in an overall yield of 75% active PDGF-AB with respect to size-exclusion chromatography and subsequent dimerization. The described approach allows renaturation at high protein concentrations and circumvents aggregation which is observed when refolding is carried out by dilution.
Müller
C
C
GBF National Research Center for Biotechnology, Biochemical Engineering Division, Braunschweig, Germany.
Rinas
U
U
D002850:Chromatography, Gel / Q000379:methods*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D002479:Inclusion Bodies / Q000737:chemistry*; D010982:Platelet-Derived Growth Factor / Q000737:chemistry*; D020673:Protein Renaturation; D011995:Recombination, Genetic; D013050:Spectrometry, Fluorescence
D010982:Platelet-Derived Growth Factor; C095477:platelet-derived growth factor AB