Recombinant hexa-His-tagged pro-HNP-1 ( Fig. 1 ) was expressed at approximately 2 mg/L in E. coli cells. The imidazole eluant from the nickel affinity column (Ni-eluant) contained a major band of ,12 kDa, consistent with the molecular weight of His 6 -proHNP1 (theory = 12647 A.M.U.; Fig. 2A ). C18 reversed phase high performance liquid chromatography (RP-HPLC) of the Ni-eluant produced two major peaks (A and B; Fig. 2B ). On reducing SDStricine PAGE, peaks A and B contained the ,12 kDa band (Fig. 2C ) and both were detected with anti-His 6 antibody confirming the presence of His 6 -proHNP1 (Fig. 2D) . On AU-PAGE, His 6 -proHNP1 present in peak A (proHNP1A) migrated more rapidly than that contained in peak B (proHNP1B; Fig. 2E ), suggesting that proHNP1A and proHNP1B are differently folded forms of the recombinant protein. This was confirmed by the finding that DTTreduced proHNP1A and proHNP1B co-eluted on RP-HPLC (data not shown) and by demonstrating that folding of proHNP1B in the presence of reduced/oxidized glutathione led to the formation of proHNP1A (Text S1 and Figure S1 ). Thus, proHNP1A and proHNP1B contain folded and misfolded His 6 -proHNP1 respectively. ProHNP1A and proHNP1B-containing fractions were pooled separately and purified to homogeneity by a second round of RP-HPLC (Fig. 2F ).