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Proopiomelanocortin (POMC), a precursor protein of ACTH, serves as the source of a large number of biologically active peptide hormones, including MSHs, CLIP, LPH, and endorphin. POMC is sequentially processed at pairs of basic amino acid residues via a sequence of enzymatic steps in a tissue-specific manner by pro-hormone convertase 1 and 2 (PC1/2) [1]. However, little is known regarding its own role or the machinery associated with its processing in detail, based on its tertiary structure.To obtain structural information related to the mechanism associated with the processing of POMC, recombinant POMC was over-expressed in E. coli cells using the artificial gene encoding POMC which was optimized at the codons and GC contents [2]. Recombinant POMC was readily over-expressed as a soluble protein and purified using several types of chromatography, such as Ni-chelate affinity and hydroxyapatite chromatography. Circular dichroism (CD) measurements of the purified POMC were carried out under several different conditions, including pH and different solvents. The results suggested that recombinant POMC possesses less α-helical and β-sheet structure in aqueous solutions used in our experiments. However, the contents of the α-helical structure of POMC were increased in a concentration dependent manner when trifluoroethanol was used as the solvent. The thermodynamic stability of POMC was also evaluated under several conditions by CD measurements. The collective results will be discussed.References[1] Seidah, N.G., Benjannet, S., Hamelin, J., Mamarbachi, A.M., Basak, A., & Marcinkiewicz, J. (1999) Ann NY Acad Sci,885, 57–74.[2] Konogami, T., Watanabe, K., Shimamoto, S., Basak, A., and Hidaka, Y. (2012) Peptide Science 2011, 367–368.

Proopiomelanocortin (POMC), a precursor protein, serves as the source of numerous biologically active peptides, including MSHs, ACTH, CLIP, LPH, and β-endorphin. POMC is processed at pairs of basic amino acid residues in a tissue-specific manner by pro-hormone convertase 1 and 2 (PC1/2). However, little is known concerning its own role and the mechanism associated with its processing, based on its tertiary structure. In addition, little information is available concerning the relation between the tertiary structures of the mature peptides and POMC.In order to obtain structural information related to the processing mechanism of POMC, recombinant POMC was previously expressed using a T7-promoter system in E. coli cells and the purified POMC was crystallized. However, the contaminating proteins affected the formation of POMC crystals. The expression level of POMC in E. coli cells was quite low, which made it difficult to eliminate contaminating proteins. Therefore, a system that permits the expression of much higher levels of the protein is required for the structural analysis of POMC.For this purpose, the chemically synthesized cDNA encoding POMC, optimized at the codons and GC contents, was used to overcome the codon bias of E. coli. The PCR-amplified cDNA of POMC was introduced into several different expression vectors, such as pET17b and pPal7, and expressed in several types of E. coli cells. The recombinant POMC was well over-expressed in SHuffle T7 E. coli cells and purified by a combination of Ni-affinity and hydroxyapatite chromatography. The results will be discussed in this paper.