Cloning and Expression of Dynorphin-32 in E. coli and Analgesic Tests in Mice Models

ABSTRACT

Chronic severe pain is most destructive to the human psyche. Presently available analgesics are effective in the treatment of acute pain, although even in this setting “analgesic gaps” remain. But for chronic pain, there is a need to develop better therapeutic strategies and analgesics than are currently available. Epidemiologic data indicates that even in prosperous, developed nations, 40 % of patients with chronic pain are only partially satisfied and 15 % are not satisfied at all with the treatments available to them. At the same time, proper treatment of pain is increasingly in demand as a human right by patients, their families, and governments. Therefore, modern medicine urgently needs more effective treatments for pain. Therefore, the challenge of this decade is to produce small molecules which mimic chemical drugs, in order to overcome the ineffectiveness of currently available analgesic drugs. This work includes identification of target receptors, selection of potent existent drugs, modelling, and selection of peptide based therapeutic drugs.METHODSThe selected mutant sequences of analgesic peptides were codon optimized using DNA 2.0 software for maximum expression in Saccharomyces cerevisiae and E. coli. Codon optimization was carried out at above 15% threshold and as per the codon usage frequency of standard Saccharomyces and E. coli tables. For yeast Alpha F mating tag and for E. coli ompA tag was used for efficient extracellular recombinant expressions. The expression studies were carried out initially in E. coli with analgesic peptides. Lac promoters were used for induction of the genes of these analgesic peptides. These peptides were also precipitated using acetone and used for the analysis. Partially purified analgesic peptides by salt and acetone precipitation were used for bioassays by Tail immersion and hot plat assays.RESULTSPositive confirmed constructs were transformed in BL21 for expression. Dynorphin recombinants in BL21 were used for expression studies. The expression of recombinant protein was confirmed by 16% SDS PAGE. The over expressed peptide was first precipitated by ammonium sulphate and dialyzed. The dialyzed samples were used for bioassay studies. Fentanyl was used as positive control and uninduced culture broth as negative control. Among the three peptides studied JV5 (Peptide F) showed 62% central analgesic activity (observed by tail immersion assay) when compared to fentanyl by oral route of administration and fentanyl treatment (hot plate assay) indicating similar action suggesting usage of this peptide as strong analgesic molecule for future pain treatments.