Magnehis protein purification system

HliR was amplified from the H. ochraceum genome by PCR using high-fidelity PrimeSTAR Max DNA polymerase (Takara Bio Inc.) and the primers HliR Exp Infu f/HliR Exp Infu r, and subsequently cloned into BamHI/XhoI double-digested pET32a by using an In-Fusion^® HD Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA). The purified plasmid was transformed into Single Step (KRX) Competent Cells (Promega, Madison, WI). For the expression of HliR, when the OD₆₀₀ reached 0.6, a final concentration of 0.1% rhamnose and 0.4 mM IPTG were added to induce protein expression. After induction for additional 20 h at 20 °C, the cells were harvested by centrifugation (6000 rpm, 5 min) and resuspended in 3 mL of extraction buffer (50 mM Tris-HCl, 0.4 M NaCl, pH 7.8). The resulting suspension was lysed by using a sonication homogenizer (50 W, five cycles) in the presence of benzonase nuclease (Novagen, Madison, WI) over ice for 30 min. The lysate was centrifuged (6000 rpm, 5 min) to remove insoluble cell debris. The crude protein extracts were stored at −30 °C for subsequent purification. A MagneHis^TM Protein Purification System (Promega, Madison, WI) was used to purify the His₆-tagged protein. The crude extract and purified HliR were analysed by SDS-PAGE (10% Tris-HCl gel) (Supplementary Fig. S19).

To express the recombinant proteins, the abovementioned plasmids were introduced into protease-defective and T7 RNA polymerase-containing E. coli strain BL21 (DE3). BL21 (DE3) cells transformed with pET32a (+) served as the control. E. coli BL21 (DE3) cells harboring MT genes in the pET32a (+) vector were grown at 37 °C overnight in the presence of 100 mg·L⁻¹ ampicillin. The cultures were then transferred to fresh Lysogeny broth (LB) medium (1%, v/v) and grown to an Optical density (OD) 600 of 0.6 to 0.8. When this OD was attained, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the culture medium at a final concentration of 1 mM, and the culture was grown at 37 °C for 3 h. The cells were used for protein purification and immunoblot analysis (data not shown). The four metallothionein-like proteins were purified using the MagneHis^TM Protein Purification System (Promega, Shanghai, China). The four fusion proteins in the sample buffer (5% ME, 2% SDS, and 62.5 mM Tris-HCl, pH 6.8) were heated at 100 °C for 10 min and separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

The recombinant Amwaprin protein and anti-Amwaprin antibody produced in our previous study [18 (link)] were used. Recombinant Amwaprin was produced in baculovirus-infected insect cells using a baculovirus expression vector system. The insect cell line Spodoptera frugiperda 9 (Sf9; Gibco BRL, Gaithersburg, MD, USA) was cultured in TC100 medium (Gibco BRL) supplemented with 10% fetal bovine serum (FBS, Gibco BRL) at 27 °C. Amwaprin cDNA was inserted into the baculovirus expression vector pBacPAK8 (Clontech, Palo Alto, CA, USA). Recombinant Amwaprin baculoviruses were infected into Sf9 cells, and the recombinant Amwaprin, including a hexahistidine tag (His-tag), was purified using a MagneHis^TM Protein Purification System (Promega, Madison, WI, USA). The recombinant Amwaprin was quantified using a protein assay kit (Bio-Rad, Hercules, CA, USA). An anti-Amwaprin antibody against the recombinant Amwaprin protein generated in BALB/c mice (Samtako Bio Korea Co., Osan, Republic of Korea) was used.