Expressing rHWTX-I in the yeast system Pichia pastoris was also attempted. Four additional amino acid residues were attached to the N-terminal of the expressed rHWTX-I, and the bioactivity of the expressed peptide was only 70% in comparison with that of the natural toxin. A baculovirus system was also used for the expression of rHWTXI, but neither the yield nor the cost was satisfactory, despite the fact that the expressed peptide demonstrated natural bioactivities. In summary, no efficient system has been developed thus far to express rHWTX-I in a way that maintains natural activities with a satisfactory yield. It has been estimated that there are more than 1 million currently existing spider species. Based on a conservative estimate, the potential number of unique spider venom peptides could be more than 12 million. Spider venoms, as well as venoms from snakes, frogs, scorpions, sea anemones, and cone snails, have been widely studied in past decades and have led to the discovery of a large number of proteins cytometry bioactive peptides. The biological diversity of animal venom peptides refined by the evolutionary process makes them preoptimized molecules that could be readily used in either structure/function studies of ion channels/receptors, or the development of modern drugs. It was proposed recently that venomics, a high-throughput approach based on a combination of MS and molecular biology methods, can be used as a new paradigm for venom exploration. However, for each and every unique bioactive peptide identified by venomics or other cutting-edge technologies, a complete structural/functional characterization is necessary before it can be used either as a research tool or a drug model molecule. In these cases, the production of a sufficient quantity of the peptide remains the greatest bottleneck. There are three major strategies of venom peptide production: classical biochemical preparation, direct peptide synthesis, and the expression of peptide-coding nucleotides. Since most venomous animals are of very small size, the utilization of bioassay-guided fractionation technologies is greatly confined due to the limited amount of venom available. It is also notable that most bioactive peptides identified from invertebrates comprise about 15 to 70 amino acid residues and are reticulated by several disulfide bridges. Therefore, the direct synthesis of these peptides is challenged by not only low efficiency and high cost, but also the oxidative folding of disulfide-rich peptides. Various systems were used previously for the recombinant expression of small peptide toxins with disulfide bridges, including bacterial systems , yeast, and cultured insect cells. Although it is hard to predict which system is suitable for the expression of a specific bioactive peptide, the E. coli system, due to its ease of handling and reasonable product/cost ratio, is always the first choice for most researchers. As mentioned above, recombinant HWTX-I was expressed in the cytoplasm of E. coli cells in fusion with GST. Although the original yield of the fusion protein was more than 10 mg/L culture, the rHWTX-I released by enzyme digestion exhibited very low bioactivity. Mass spectrometry analysis demonstrated that no disulfide bridge formed, yet the formation of the three disulfide bonds in the natural form is critical to HWTX-I��s functions. It was reported that the cytoplasm of E. coli cells contains high levels of reduced glutathione ; therefore, the potential of the cytoplasm is too reducing for most disulfide bonds to form.