This work reports a new approach to clone active Neopladine 2 (Neo2), an anticancer peptide from Tityus discrepans scorpion venom. RNA was extracted from the venom gland and cDNA was obtained by RTPCR using complementary primers for the nucleotide sequence coding native Neo2 (nNeo2) N-terminal. Deduced amino acid sequence shows that Neo2 is comprises by 245 residues. The best conditions to achieve active Neo2 expression in Escherichia coli were 16 degrees C for 20-24 h. Active recombinant Neo2 (rNeo2) was obtained as inclusion bodies (IBs) with a yield of 3.5 mg/L culture. rNeo2 purification by HPLC revealed eleven isoforms with differences in polarity and activity. Only rNeo2a and rNeo2k isoforms induces cancer cells death like nNeo2. rNeo2a, rNeo2g and rNeo2j were the most interesting isoforms because their apoptosis/necrosis (A/N) index and their yield. Cloned isoforms have attractive properties because some of them had higher activity than the native protein, which can be exploited for biotechnological and biomedical applications. The in silico Neo2 3D structure predicted a globular protein, with five alpha-helixes and four beta-sheet maintained by 4 sulfide pairing, highly polar, with charged amino acids in its surface and two faces with different electrostatic potentials, suggesting an important dipolar moment. (C) 2016 Published by Elsevier Ltd.