Pd 10 column chromatography

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.

Tat-CIAPIN1 protein was prepared as described previously [22 (link)]. To obtain Tat-CIAPIN1 and CIAPIN1 protein, the cDNA for human CIAPIN1 was amplified by PCR and the product was cloned into Tat expression vector. CIAPIN1 protein, without the Tat peptide, was also prepared to use as a control. Then, the Tat-CIAPIN1 and CIAPIN1 protein was expressed in Escherichia coli BL21 (DE3) cells by adding 0.5 mM isopropyl-β-D-thiogalactoside (Duchefa, Haarlem, Netherlands). Subsequently, Tat-CIAPIN1 protein was purified by a Ni²⁺-nitrilotri-acetic acid Sepharose affinity column (Qiagen, Valencia, CA, USA) and PD-10 column chromatography (Amersham, Braunschweig, Germany) according to the manufacturer’s instructions. Purified Tat-CIAPIN1 and CIAPIN1 protein concentration was determined by the Bradford assay [68 (link)].

After the plasmids were transformed into E. coli BL21 (DE3) cells, the PEP-1-PON1 and control PON1 proteins were induced by adding 0.5 mM isopropyl-β-D-thio-galactoside (Duchefa, Haarlem, Netherlands) at 37°C for 6 h. Recombinant proteins obtained from harvested cells were lysed by sonication after which the proteins were purified by Ni²⁺-nitrilotriacetic acid Sepharose affinity column chromatography (Qiagen, Valencia, CA, USA) and PD-10 column chromatography (Amersham, Brauncschweig, Germany) according to the manufacturer’s instructions [28] (link)–[36] (link). To remove endotoxins of proteins, purified control PON1 and PEP-1-PON1 were treated with a Detoxi-Gel™ endotoxin removing gel (Pierce, Rockford, IL, USA) as per manufacturer’s instructions [34] (link). The Bradford assay was used to estimate protein concentration [37] (link).