Ampicillin

GST-tagged recombinant proteins were expressed in E. coli BL21 transformed with pGEX-3X fusion constructs. For each protein, an aliquot of an overnight pre-culture was used to inoculate a fresh complex medium (LB) containing 50 μg/ml Ampicillin (Serva) and maintained at 37 °C up to mid-log phase (A₆₀₀ = 0.4–0.6). Cultures were then incubated at 30 °C for 3 h with IPTG (1 mM) for induction. Bacterial cells were harvested and washed twice in cold PBS, before being resuspended in PBS (replaced by distilled water when samples were prepared for insect bioassays) and added with 1 ml acid-washed glass beads (710–1180 μm) (Sigma-Aldrich) for cell breaking through 5 cycles of vortexing (20 s at 100 W) and cooling in ice (20 s). Lysates were then centrifuged at 5,000 rpm for 30 min at 4 °C, and the resulting pellets and supernatants were stored at −20 °C for further use, after being quantified for protein content by Bio-rad Protein assay (Bio-rad).
GST-tagged proteins were purified from lysates following the Glutathione Sepharose 4 Fast Flow one-step protocol (GE Healthcare) according to manufacturer’s instructions, which allowed to collect different protein eluates.
The result of each expression and purification step was checked by SDS-PAGE or Western Blot analysis.

Quantification of missense and WT mRNA was done with total RNA of EHTs and of myectomy tissue received from the index patient. RNA (200 ng) was reverse‐transcribed to cDNA according to the instructions of the manufacturer's protocol (SuperScript™ III First‐Strand Synthesis System, Invitrogen). cDNA was amplified with PrimeStar^® HS DNA Polymerase in a 50‐μl PCR approach, for 35 cycles according to the instructions of the manufacturer's protocol (Primers, Appendix Table S3). Generated PCR fragments were purified with the QIAquick PCR Purification Kit (Qiagen) and cloned with the CloneJET PCR Cloning Kit (Thermo Fisher Scientific) for discrimination of allele‐specific mRNA transcripts. Single colonies of transformed One Shot^® TOP10 E. coli were transferred to overnight cultures [5 ml, lysogeny broth (BD), ampicillin (100 μg/ml; Serva)]. Plasmid extraction (NucleoSpin^® Plasmid, Macheray‐Nagel) was performed the next day and send for sequencing (Eurofins Genomics).

Ectoine and 5-hydroxyectoine were kindly provided by the bitop AG (Witten, Germany). Anhydrotetracycline-hydrochloride (AHT) was purchased from IBA GmbH (Göttingen, Germany). Acetonitrile (HPLC-grade) was obtained from VWR International GmbH (Darmstadt, Germany). Ampicillin and all other chemicals were purchased from Serva Electrophoresis GmbH (Heidelberg, Germany) and Carl Roth GmbH (Karlsruhe, Germany). Radiolabeled [1-¹⁴C]glycine betaine (55 mCi mmol⁻¹) was bought from American Radiolabeled Chemicals Inc. (St. Louis, MO; USA).