L selenomethionine

The DNAs encoding pZIP4-ECD (Genebank code: ELK11751, residues 36–322) and pZIP14-ECD (UniProtKB/Swiss-Prot: A5D7L5, residues 61–174) were synthesized (Genescript Inc.) with optimized codon for E. coli expression, and subcloned into a pLW01 vector (gift from Dr Lucy Waskell at University of Michigan). The DNA sequence of full-length pZIP4 was optimized for human cell expression and also synthesized by Genescript Inc. The complementary DNA of human ZIP4 from Mammalian Gene Collection was purchased from GE Healthcare (GenBank code: BC062625). Both hZIP4 and pZIP4 were subcloned into a modified pEGFP-N1 vector (Clonetech) in which the downstream EGFP gene was deleted and an HA tag was inserted before the stop codon. All the mutations were made using QuikChange mutagenesis kit (Agilent). The sequences of the primers used in the work are listed in Supplementary Table 1. L(+)-Selenomethionine was purchased from Acros Organics. The reagents for protein crystallization were obtained from Hampton Research. Thrombin was purchased from Novagen.

The sequence of the full-length Mot1 (1 – 1886) was isolated from the Chaetomium thermophilum cDNA library and cloned into pETDuet-1 vector (Novagen, Germany) harboring N-terminal His₆ tag followed by TEV cleavage site. CtMot1^ΔC(1 – 1836) was cloned into pET21 vector containing PreScission protease cleavage site and C-terminal His₆ tag. Both constructs were expressed in Escherichia coli Rosetta(DE3) cells (Novagen) and purified using Ni²⁺-NTA agarose (QIAGEN, Germany). After proteolytic cleavage of the expression tags, the proteins were further purified using ion-exchange chromatography (HiTrap Q HP, GE Healthcare, Germany) and size exclusion chromatography (HiLoad 16/60 200 pg, GE Healthcare). Proteins were concentrated to ~15 mg/ml in 20 mM Tris pH 7.5, 50 mM NaCl and 15% glycerol and stored at −80°C. Selenomethionine labelling of CtMot1^∆C was performed in E. coli B843 (Novagen) using SelenoMethionine Medium Base and Nutrient Mix (Molecular Dimensions, UK) supplemented with L(+)-Selenomethionine (Acros Organics, Germany) at 42 mg/L. Purification of selenium-derivatized protein was performed according to the same protocol as for the native protein.