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Published on: February 12, 2019
The refolding of different alpha-fetoprotein variants
Susanna S J Leong1, Anton P J Middelberg
1Centre for Biomolecular Engineering, The University of Queensland, St. Lucia, Australia. ssjl2@cam.ac.uk
Alpha-fetoprotein (AFP) foldability is independent of glycosylation, fatty acid binding, or refolding method. This study demonstrates AFP denaturation is reversible, refolding correctly in a redox-controlled environment.
Area of Science:
- Biochemistry
- Protein Chemistry
- Molecular Biology
Background:
- Alpha-fetoprotein (AFP) is a crucial biomarker, but its refolding properties, especially concerning glycosylation, remain incompletely understood.
- Previous studies suggested AFP denaturation might be irreversible and dependent on fatty acid binding.
Purpose of the Study:
- To investigate the impact of glycosylation on AFP foldability and refolding.
- To determine if AFP denaturation is reversible under specific conditions.
- To explore the role of fatty acid binding in achieving a rigid AFP tertiary structure.
Main Methods:
- Comparative analysis of refolding for glycosylated and nonglycosylated AFP variants using reversed-phase HPLC and circular dichroism spectroscopy.
- Refolding of inclusion body-derived AFP via dialysis and dilution under redox-controlled conditions.
- Enzyme-linked immunosorbent assay (ELISA) to assess bioactivity of refolded AFP.
Main Results:
- Both glycosylated and nonglycosylated AFP variants refolded successfully with comparable yields and spectroscopic profiles.
- Refolding of inclusion body-derived AFP confirmed its reversibility and structural integrity.
- AFP refolding efficiency was sensitive to inclusion body contaminants, improving with purity.
- Refolded AFP variants demonstrated bioactivity via ELISA.
Conclusions:
- AFP foldability is independent of glycosylation, fatty acid presence, and refolding method (dialysis or dilution).
- AFP denaturation is reversible when refolded in a redox-controlled environment promoting disulfide shuffling.
- Fatty acid binding is not essential for forming a rigid AFP tertiary structure.
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