High-Performance Reversed-Phase Flash Chromatography Purification of Peptides and Chemically Modified Insulins.

Preparative reversed-phase HPLC is the established method for the purification of peptides, but has significant limitations. We systematically investigated the use of high-performance reversed-phase flash chromatography (HPFC) to rapidly purify laboratory-scale quantities of crude, synthetic peptides and chemically modified insulins. We demonstrated these methods for a diverse set of peptides, including short, medium, and long peptides. Depending on the purity profile of the peptide, HPFC can be used either as the sole purification method, or as a pre-purification method prior to final HPLC purification. Furthermore, HPFC is suitable for the purification of peptides that are not fully in solution. We provide guidelines for the HPFC of synthetic peptides and small proteins, including the choice of columns, eluents, and gradients. We believe that HPFC is a valuable alternative to HPLC purification of peptides and small proteins.

Identification of putative insulin-like peptides and components of insulin signaling pathways in parasitic platyhelminths by the use of genome-wide screening.

No endogenous insulin-like peptides in parasitic flatworms have been reported. Insulin receptors from flukes and tapeworms have been shown to interact directly with the host-derived insulin molecule, which suggests the exploitation of host-derived insulin. In this study, a strategy of genome-wide searches followed by comprehensive analyses of strictly conserved features of the insulin family was used to demonstrate the presence of putative insulin-like peptides in the genomes of six tapeworms and two flukes. In addition, whole insulin signaling pathways were annotated on a genome-wide scale. Two putative insulin-like peptide genes in each genome of tapeworms and one insulin-like peptide gene in each genome of flukes were identified. The comprehensive analyses revealed that all of these peptides showed the common features shared by other members of the insulin family, and the phylogenetic analysis implied a putative gene duplication event in the Cestoda during the evolution of insulin-like peptide genes. The quantitative expression analysis and immunolocalization results suggested a putative role of these peptides in reproduction. Entire sets of major components of the classic insulin signaling pathway were successfully identified, suggesting that this pathway in parasitic flatworms might also regulate many other important biological activities. We believe that the identification of the insulin-like peptides gives us a better understanding of the insulin signaling pathway in these parasites, as well as host-parasite interactions.