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Preparative Scale Production of Recombinant Human Transthyretin for Biophysical Studies of Protein-Ligand and Protein-Protein Interactions

AuthorsCotrina, Ellen Y.; Vilà, Marta; Nieto, Joan; Arsequell, Gemma ; Planas, Antoni
Recombinant expression
Fed-batch culture
Protein yield
Protein-ligand interactions
Protein-protein interactions
Amyloid diseases
Generalitat de Catalunya
Fundació La Marató de TV3
Ford España
Issue Date2020
PublisherMultidisciplinary Digital Publishing Institute
CitationInternational Journal of Molecular Sciences 21(24): 9640 (2020)
AbstractHuman transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aβ binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-β (Aβ) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aβ interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.
Description© 2020 by the authors.
Publisher version (URL)https://doi.org/10.3390/ijms21249640
Appears in Collections:(IQAC) Artículos
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