title: Production and characterization of neurosecretory protein GM using Escherichia coli and Chinese Hamster Ovary cells.
abstract: Neurosecretory protein GL (NPGL) and neurosecretory protein GM (NPGM) are paralogs recently discovered in birds and in mammals. The post-translational products of NPGL and of NPGM genes include a signal peptide sequence, a glycine amidation signal, and a dibasic amino acid cleavage site. This suggests that the mature forms of NPGL and of NPGM are small proteins secreted in the hypothalamus and containing an amidated C-terminus. However, endogenous NPGL and NPGM have not yet been identified. Chicken NPGL and NPGM have two highly conserved Cys residues that are likely to form a disulfide bond, while mammalian NPGM has one additional Cys residue located between the two conserved Cys residues and the correct disulfide bond pattern is unclear. In this study, we prepared rat NPGM to elucidate the structure of its mature form. We first expressed the predicted mature NPGM, containing an extra C-terminal Gly, in Escherichia coli SHuffle cells, which are engineered to promote the formation of native disulfide bridges in recombinant proteins. We observed the presence of a disulfide bond between the N-terminal Cys residue and the second Cys residue, while the C-terminal Cys residue was free. Secondly, we transfected a construct containing the entire NPGM open reading frame into Chinese Hamster Ovary cells, and observed that NPGM was cleaved immediately after the signal peptide and that it was secreted into the medium. Furthermore, the protein presented a disulfide bond at the same location observed in recombinant NPGM.
authors.affiliation: Section of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Masuda
authors.affiliation: Section of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
authors.forename: Megumi
authors.initials: M
authors.lastname: Furumitsu
authors.affiliation: Section of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
authors.forename: Shusuke
authors.initials: S
authors.lastname: Taniuchi
authors.affiliation: Section of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
authors.forename: Eiko
authors.initials: E
authors.lastname: Iwakoshi-Ukena
authors.affiliation: Section of Behavioral Sciences, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan.
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ukena
keywords: CHO cells, Chinese Hamster Ovary cells; Chinese Hamster Ovary cell; Disulfide bond; E. coli, Escherichia coli; Escherichia coli; Free cysteine residue; NPGL, neurosecretory protein GL; NPGM, neurosecretory protein GM; Neurosecretory protein GL; Neurosecretory protein GM; SP, signal peptide; TF, trigger factor