, 2011). The crude synthetic peptide was dissolved at a protein concentration of 6 mM in 0.1 M Tris buffer pH 7.5 and then reduced with

20 mM find more DTT at RT for 1hr. The reduced peptide was added to the folding solution containing 0.1 M Tris buffer pH 7.5 and a mixture of 0.15 mM Cysteine and 1.5 mM Cystine at a final peptide concentration of 24.5 μM. Refolding and formation of the correct disulphide bridging pattern was achieved during 2 days at 40 °C. The refolded synthetic toxin was purified by reversed-phase HPLC on a semi-preparative C18 column (Phenomenex Jupiter, USA) using a 35 min gradient from 23% to 45% of 60% acetonitrile in 0.1% TFA. Refolding was confirmed by MALDI-TOF MS and bioassay. A chromatographic comparison of synthetic GTX1-15 with the samples of native, folded and reduced peptides by RP-HPLC (Shimadzu, Japan) using an analytical C18 column (Phenomenex, Kinetex, USA). RP-HPLC analysis was achieved within a 10 min linear gradient from 5% to 60%

acetonitrile (Fig. 2D, left). GsTx1-15 elutes in these conditions as a double peak on the HPLC chromatogram once in its native form or refolded synthetic form (which both contain 3 disulfide bridges as detected by MS analysis, see Section 3.2 for details), while eluted as a single peak in the reduced synthetic form. MS analysis of the two peaks show no detectable differences (data not shown) and the fact that the native and synthetic peptides behave in a very similar way suggests that the peptide is homogenous. In a recent paper (Chunxiao et al., 2011) describing Ku-0059436 supplier the synthesis of a mature protein, the authors have also observed a homogenous protein eluting as two

peaks in HPLC chromatograms, depending also on the solvents used. Crude peptide was weighted, dissolved in water and measure at 280 nm. The reducing of the peptide was carried out by DTT which was added to a final concentration of 20 mM and incubated at RT for 1 h. The reduced peptide was subjected to oxidative folding reaction in a 2 M Ammonium acetate buffer (pH = 7.0) containing 1 mM GSH, 0.1 mM GSSG and 1 mM EDTA. The reduced peptide was added to the solution drop wise in 6 portions to a final concentration of 10 μM. The solution was stirred at 24 °C for 120 h. The refolded material was purified tetracosactide by a three-step purification procedure, containing a RP-HPLC and further ion-exchange chromatography: The RP-HPLC purification was carried out using Jupiter C18 column (Phenomenex, USA) by liner gradient using 60% acetonitrile containing 0.1% TFA as buffer B. The peak fractions were joined and lyophilized. Excess contamination were removed by ion-exchange chromatography using Luna SCX column (Phenomenex, USA) by 25 min liner gradient of 700 mM potassium chloride in potassium phosphate buffer (pH = 2.5) containing 25% acetonitrile as buffer B.