κ-Hefutoxin1, a novel weak potassium-ion-channel toxin present in the venom of the scorpion Heterometrus fulvipes, is a 22-residue peptide which has a unique spatial fold consisting of two parallel helices linked by two disulfide bridges without any β-sheets. In order to evaluate the structural contribution of the disulfide bonds, wild and three mutant forms of κ-Hefutoxin1 were cloned, expressed, purified and structurally analyzed. To do so, synthetic genes encoding wild and three mutant forms of k-Hefutoxin1 were designed using appropriate codons and synthesized using overlapping primers. In the mutant forms, alternative pairs of cystein residues, which participate in the formation of disulfide bonds, were replaced by serine (Mut1: C4S, C22S; Mut2: C8S, C18S; Mut3: C4S, C22S, C8S and C18S). To facilitate cloning into expression vector, EcoRI and BamHI restriction sites were inserted to the flanking ends of the genes. Moreover, Tev cleavage site was added to the N-terminal part of the genes. The amplified k-Hefutoxin1 genes of wild and mutant forms were cloned into pET32a vector, followed by transformation into E.coli host strain DH5α. The positive colonies with recombinant plasmid were first screened by PCR analysis and finally confirmed by sequencing. Then, the correct recombinant plasmid was transformed into E.coli host strain BL21 in which protein expression was induced by IPTG. After assuring protein expression by polyacrylamide gel electrophoresis, the cells containing wild and mutant forms of k-Hefutoxin1 were lysed by sonication. Obtained proteins were finally purified and structurally analyzed by CD spectroscopy and NMR. In this study, we were able to ascertain the effect of absence of one or both of the disulfide bonds on the structure of k-Hefutoxin1.