Preparative Scale Production of Recombinant Human Transthyretin for Biophysical Studies of Protein-Ligand and Protein-Protein Interactions.
Int J Mol Sci
; 21(24)2020 Dec 17.
Article
en En
| MEDLINE
| ID: mdl-33348885
ABSTRACT
Human 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 Aß binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-ß (Aß) 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 TTRAß 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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes
/
Prealbúmina
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Bibliotecas de Moléculas Pequeñas
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Amiloide
Límite:
Humans
Idioma:
En
Revista:
Int J Mol Sci
Año:
2020
Tipo del documento:
Article
País de afiliación:
España