Expression and Purification of Tat-PDIA3 Protein

A Tat expression vector was prepared as described in the previous study.^{43 (link)} To construct a Tat-PDIA3 protein, PDIA3 cDNA was amplified by polymerase chain reaction (PCR) with the use of the following primers: sense primer 5′-CTCGAGATGCGCCTCCGC-3′ and antisense primer 5′-GGATCCTTAGAGATCCTCCTGTGCC-3′. After subcloning the PCR product into a TA cloning vector, it was ligated into the Tat expression vector. A PDIA3 expression vector without the Tat-protein transduction domain was also constructed to be used as a control (Control-PDIA3).
The Tat-PDIA3 and control-PDIA3 plasmids were expressed in Escherichia coli BL21 cells, which were treated with 0.1 mM isopropyl-β-d-thiogalactoside (IPTG; Duchefa, Haarlem, Netherlands) at 18 °C for 8 h, and purified using a Ni²⁺-nitrilotriacetic acid Sepharose affinity column and PD-10 column chromatography (Amersham, Braunschweig, Germany) according to the manufacturer's instructions. The purified proteins were treated using Detoxi-Gel™ endotoxin removing gel (Pierce, Rockford, IL, USA) to remove endotoxins. Endotoxin levels in the proteins were below the detection limit (<0.1 EU/ml) as tested using a Limulus amoebocyte lysate assay (BioWhittaker, Walkersville, MD, USA). The Bradford assay^{44 (link)} was used to estimate the quantity of purified Tat-PDIA3 and control-PDIA3 protein.