E coli shuffle t7 cells

RBD (UniProt ID P0DTC2) with Cys538Ala was expressed in SHuffle T7 E. coli cells (New England Biolabs, Ipswich, MA, USA), using a pET15b expression vector in LB medium containing 50 μg/mL ampicillin as previously reported [10 (link)]. The protein was purified by using denaturing nickel–nitrilotriacetic acid (Ni-NTA) (Wako, Tokyo, Japan) chromatography and reverse-phase (RP) high-performance liquid chromatography, lyophilized and stored at −30 °C until use [17 (link)].

Plasmids encoding BECN1-fused knob domains were transformed into chemically competent SHuffle T7 E. coli cells (New England Biolabs) and plated onto LB-agar plates supplied with 50 µg/ml of kanamycin. The colonies were transferred into 2xTY, 50 µg/ml kanamycin and incubated at 37°C for 16 h. The resulting cultures were used to inoculate 3 L of fresh medium. Recombinant expression was induced by adding 1 mM IPTG at OD₆₀₀ ~ 0.6, and the temperature was decreased to 18°C. After 16 h, the cells were harvested by centrifugation (7,000 g for 1 h), resuspended in PBS, 0.5 M NaCl, 4 M guanidinium chloride and lysed by passing through a French press. The lysate was cleared by centrifugation (20,000 g for 1 h), passed through 0.22 µm filters and loaded onto a HisTrap HP column (Cytiva) equilibrated with the lysis buffer. Bound proteins were refolded by running a linear 4-0 M guanidinium chloride gradient. After a wash with PBS, 0.5 M NaCl, 20 mM imidazole, the knob domains were eluted by PBS, 0.5 M NaCl, 200 mM imidazole. Pooled fractions containing the protein were purified by reversed-phase (RP) HPLC as described in (38 (link)). The product was lyophilised and stored at −20°C for subsequent analyses.

The AMN T41I, L59P, M69K, C234F and G254E mutant constructs were generated with the In-Fusion HD cloning kit (Clontech) using the AMN(20–357)–cubilin(26–135) pACYCDuet-1 vector as template (Supplementary Table 1). The AMN–cubilin pACYCDuet-1 vector was transformed into ShuffleT7 E. coli cells (New England Biolabs Inc.). Cell cultures were grown in 2xTY growth medium with 34 µg ml⁻¹ chloramphenicol and induced overnight at 20 °C with 1 mM IPTG. The cells were resuspended 20 mM HEPES pH 7.6, 500 mM NaCl, 5 mM Imidazole, 10% glycerol (lysis buffer) and lysed by sonication. The lysate was centrifuged at 30,000×g for 20 min and the supernatant loaded on a 5 ml cOmplete^TM His-Tag Purification Column (Roche) equilibrated in lysis buffer. The protein was eluted by running a linear gradient from 5 to 500 mM imidazole.