Preparation of a Orexin Precursor Protein by Chemical Digestion

Abstract

Endogenous peptide hormones are often produced in vivo in the form of precursor proteins that contain pre- and pro-leader sequences, which are subsequently processed into the biologically active mature peptide. Therefore, it is important to study the folding of precursor proteins to understand the mechanisms of post-translational modifications and the formation of the active conformation of the mature peptide. However, it is generally difficult to obtain precursor proteins from intact organs because only small amounts are produced in vivo. To overcome this issue, an E. coli expression system is employed to prepare recombinant proteins.Pro-orexin consists of orexin A, B, and a C-terminal peptide. To investigate the mechanism responsible for the processing and post-translational modifications of orexin peptides, we tried to express the recombinant pro-orexin in E. coli cells. However, recombinant pro-orexin was not expressed in E. coli cells. In addition, it was also difficult to obtain pro-orexin, even though Trx-fused pro-orexin was readily produced in E. coli cells because the digestion of Trx-fused pro-orexin with an enzyme yielded significant amounts of non-specific digests. Therefore, to solve this issue, we employed a chemical method to release pro-orexin from fusion proteins.K. Pane et al. recently reported a chemical method to obtain a target protein from an onconase-fused (ONC-fused) protein. They applied a chemical cleavage of the fusion protein at the Asp-Pro sequence with acetic acid. We employed this chemical method to produce recombinant pro-orexin. ONC-fused pro-orexin was successfully over-expressed as an inclusion body in E. coli BL21(DE3) cells and treated with acids under several sets of conditions to cleave the Asp-Pro sequence of ONC-fused pro-orexin. The results will be discussed in this paper.