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

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 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.

Keywords: amyloid diseases; fed-batch culture; protein yield; protein-ligand interactions; protein-protein interactions; recombinant expression; transthyretin.

Figure 1

(A) Induction time for transthyretin (TTR) expression in flask cultures. SDS-PAGE of protein fractions after partial purification by anion exchange chromatography from cultures induced with IPTG. Lanes: M: molecular weight marker, 1. no IPTG induction (no TTR expression), 2. Induction at late exponential phase, 3. mid exponential phase, 4, beginning of the exponential growth. M: monomer, D: dimer. (B) MALDI-TOF MS and SDS-PAGE of purified wtTTR. (C) MALDI-TOF MS of Y78F TTR (left) and reduced Y78F TTR (right).

Figure 2

Acid-induced fibrillogenesis of Y78F TTR isoforms S-GSH (glutathionated) and free Cys (reduced with DTT). (A) Initial rate of acid-induced fibrillogenesis determined for two different protein batches (1 and 2). (B) Absorbance monitoring for 72 h. Inset. Magnification of the initial region.

Figure 3

TTR fibrillogenesis inhibitors. Reproducibility of IC₅₀ (μM) determination with different TTR Y78F protein batches. Initial rate of acid-induced fibrillogenesis at increasing concentrations of inhibitor.

Figure 4

Small-molecule chaperones of TTR:Aβ interaction. Reproducibility with two different wt TTR protein batches (1, 2). % reduction of aggregates formation (RA%) relative to Aβ(12–28) after 6 h incubation: Aβ(12–28) + TTR = 78.1 ± 2.9; Aβ(12–28) + (TTR + DIF) = 78.3 ± 2.5; Aβ(12–28) + (TTR + IDIF) = 96.5 ± 3.1; Aβ(12–28) + (TTR + tafamidis) = 77.5 ± 1.7.