Heterologous Expression and Purification of Ghlac

The gene encoding Ghlac WT (NCBI accession No.: ORJ60343.1, https://www.ncbi.nlm.nih.gov/protein/ORJ60343.1, accessed on 15 June 2019) was codon-optimized, synthesized, and cloned in the pET-28a (+) vector using the Nco I and Xho I restriction sites. The obtained recombinant vector pET28a-Ghlac-WT was transformed into E. coli BL21 (DE3). The cells containing the recombinant vector were grown in Luria–Bertani (LB) medium supplemented with 25 mg/L of kanamycin. When OD₆₀₀ reached 0.6, 0.5 mM IPTG and 0.5 mM CuSO₄ were added to induce the expression of Ghlac, and then the cells continued to grow at 16 °C for 16 h. The cells were collected by centrifugation at 4000× g for 30 min and homogenized using a JN-Mini homogenizer (JNBio, Guangzhou, China). The recombinant Ghlac in the supernatant was purified using Ni-NTA resin according to the reported method [50 (link)]. The purified Ghlac in 20 mM phosphate buffer (pH 7.4) was stored at −80 °C. The purity and molecular mass of Ghlac were assessed by SDS-PAGE. The UV/visible absorption spectrum of Ghlac was scanned in the range of 200–800 nm using a SpectraMax M2e Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The copper content of Ghlac was analyzed with an iCAP Qc inductively coupled plasma mass spectrometry (ICP-MS) (ThermoFisher Scientific, Waltham, MA, USA) [22 (link)].