The oxidation and correct formation of disulfide bonds is an important biochemical modification of many proteins. Early observations showed that disulfide bond formation proceeds much faster in vivo than in vitro, suggesting the existence of a catalyst for protein oxidative folding in living cells . Protein disulfide isomerase (PDI, EC 184.108.40.206) is a eukaryotic oxidoreductase that catalyzes the oxidation, reduction, and isomerization of disulfide bonds in nascent polypeptides . The subcellular localization and its function suggest that PDI plays a key role in the folding of proteins delivered to the secretory pathway .
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Although the key role of EhPDI as oxidoreductase enzyme has been revealed, a biochemical study has yet to be performed. So, to understand how EhPDI participates in the disulfide oxidation and rearrangement as well as in protein folding, we have analyzed these activities using well-known standard assays. Here, we report a highly reproducible method for purification of recombinant EhPDI, expressed in Escherichia coli cells, that yields high quality protein suitable for biochemical characterization. We have found that recombinant EhPDI behaves mainly as disulfide oxidase and isomerase and exhibits chaperone-like activity; moreover, these activities are inhibitable by the antibiotic bacitracin, a well-known PDI inhibitor, tempting the idea of designing specific and selective EhPDI-inhibitors.
2. Material and Methods
Taq DNA polymerase and T4 DNA ligase were from Roche, while endonucleases were from New England Biolabs. Plasmid pQE30 and Ni-NTA-agarose were from Qiagen. Hen egg white lysozyme, bovine´s insulin and ribonuclease A, as well as the MB-grade chemicals were from Sigma-Aldrich. PD10 columns were from Amersham.
2.2. Plasmid construct
The cDNA encoding the mature polypeptide of EhPDI was amplified by PCR using the plasmid pBPelB:EhPDI as template  and the synthetic oligonucleotides 5´-cggatccGCTGATGTAGTATCATTAAATC- 3´ and 5´-gggaagcttaGAAAACTTCAAGTACATT- 3´ as sense and anti-sense primers, respectively. The sense primer includes the sequence for BamHI restriction site (italized), while the anti-sense primer includes the sequence for the HindIII (italized) and the stop codon (underlined). The PCR product was digested with specific endonucleases and then subcloned in-frame into plasmid pQE30. The recombinant plasmid (pQHPDI) was initially characterized by endonuclease digestion and then by DNA sequencing.
2.3. Expression and purification of recombinant EhPDI
The recombinant EhPDI protein was expressed, in cultures of Escherichia coli XL1-Blue MRF´ (Stratagene) harboring the plasmid pQHPDI, according to the Qiagen´s QIAexpressionist™ handbook, with some modifications. Briefly, after 4 hours of induction with 0.1 mM of IPTG, the bacterial cells were harvested and lysed under denaturing conditions. The soluble lysate was slowly loaded on to an agarose-NTA-Ni column and, after metal-affinity-binding, the protein was on-column refolded by exchanging the buffers to native-oxidative conditions. Finally, the recombinant protein was eluted, collected and analyzed by SDS–PAGE. The recombinant EhPDI-enriched fractions were pooled together, loaded into a PD10 column and eluted in 20 mM Tris-HCl buffer (pH 8.0). The protein concentration was determined by the Bradford colorimetric assay, using BSA as standard, or by UV spectrophotometry at OD280, using the calculated absorptivity coefficient, 0.1% = 0.856 .
2.4. Oxidoreductase enzyme activity
Reductase assay. The disulfide reduction of bovine insulin catalyzed...