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Publication protocol
Cloning Expression and Purification of His8-PduX and Native PduX: PCR was used to amplify the pduX coding sequence from pEM55 (25). The forward primer for fusing eight histidines to the N terminus was 5′-GCCGCCAGATCTATGAAACACCATCACCATCATCACCACCATATGCGCGCACACTATTCGTACC-3′. The reverse primer was 5′-GCCGCCAAGCTTATCACTGCAGTTTGACCCCGCC-3′. The cloning procedure and the protein production and purification procedure were carried out as described previously (6). For removal of the N-terminal His8 tag and purification of the non-tagged PduX protein, we used TAGZyme DAPase Enzyme (Qiagen) following the manufacturer's instructions. This procedure produced native PduX lacking the N-terminal methionine.
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