The refolding of different alpha-fetoprotein variants.
The effect of glycosylation on AFP foldability was investigated by parallel quantitative and qualitative analyses of the refolding of glycosylated and nonglycosylated AFP variants. Both variants were successfully refolded by dialysis from the denatured-reduced state, attaining comparable "refolded peak" profiles and refolding yields as determined by reversed-phase HPLC analysis. Both refolded variants also showed comparable spectroscopic fingerprints to each other and to their native counterparts, as determined by circular dichroism spectroscopy. Inclusion body-derived AFP was also readily refolded via dilution under the same redox conditions as dialysis refolding, showing comparable circular dichroism fingerprints as native nonglycosylated AFP. Quantitative analyses of inclusion body-derived AFP showed sensitivity of AFP aggregation to proteinaceous and nonproteinaceous inclusion body contaminants, where refolding yields increased with increasing AFP purity. All of the refolded AFP variants showed positive responses in ELISA that corresponded with the attainment of a bioactive conformation. Contrary to previous reports that the denaturation of cord serum AFP is an irreversible process, these results clearly show the reversibility of AFP denaturation when refolded under a redox-controlled environment, which promotes correct oxidative disulfide shuffling. The successful refolding of inclusion body-derived AFP suggests that fatty acid binding may not be required for the attainment of a rigid AFP tertiary structure, contrary to earlier studies. The overall results from this work demonstrate that foldability of the AFP molecule from its denatured-reduced state is independent of its starting source, the presence or absence of glycosylation and fatty acids, and the refolding method used (dialysis or dilution).
Leong
Susanna S J
SS
Centre for Biomolecular Engineering, The University of Queensland, St. Lucia, Australia. ssjl2@cam.ac.uk
Middelberg
Anton P J
AP
D000818:Animals; D030801:Animals, Genetically Modified; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D005312:Fetal Blood; D006031:Glycosylation; D006041:Goats; D006801:Humans; D002479:Inclusion Bodies; D008892:Milk / Q000737:chemistry; D017510:Protein Folding; D020673:Protein Renaturation; D000509:alpha-Fetoproteins / Q000737:chemistry* / Q000235:genetics
D000509:alpha-Fetoproteins