Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues

Figure 3

Purity analysis of SOD1s by SDS-PAGE and HPLC. (a) Purity of SOD1s was analyzed by SDS-PAGE. Lane 1: molecular weight standard ladder (kDa). Lanes 2–5: purified wtSOD1 (lane 2), mhSOD1/C6S (lane 3), mhSOD1/C111S (lane 4), and mhSOD1/C6S/C111S (lane 5). (b) HPLC analysis of purified wtSOD1 (a), mhSOD1/C6S (b), mhSOD1/C111S (c), and mhSOD1/C6S/C111S (d). A reverse-phase column was used for HPLC (Beckman, 250 × 5.0 mm ID, Kromasil C18, Akzo Nobel Co. Ltd. (Sweden)). 5 μg samples in PBS were injected into the column and then eluted by linear gradient: 100% solution A (water, 5% acetone, and 0.1% TFA) to 40% solution B (acetone, 0.1% TFA) over 40 minutes with a flow rate of 1.0 mL/min. The eluted fractions were analyzed at 280 nm, and the purity of the proteins was calculated as a percentage of the total peak area.

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