title: Expression, receptor binding, and biophysical characterization of guinea pig insulin desB30: a monomeric insulin variant.
abstract: Here we report, for the first time, the heterologous expression of desB30 guinea pig insulin (GI desB30) in the yeast Saccharomyces cerevisiae. The affinities of GI desB30 for the insulin receptor A and the IGF-I receptor were also quantified for the first time. Small-angle X-ray scattering and analytical ultracentrifugation studies confirmed that GI desB30 did not form dimers or hexamers, in contrast to human insulin. Size-exclusion chromatography connected to inductively coupled plasma mass spectrometry revealed that GI desB30 has affinity towards several divalent metal ions. These studies did not indicate the formation of any larger structures of GI desB30 in the presence of various divalent metal ions, but did indicate that GI desB30 has an affinity towards Mn, Co, and Cu ions. Finally, the low affinity for the insulin receptor and the very low affinity for the IGF-I receptor by GI desB30 were quantified.
authors.affiliation: Diabetes Protein Engineering, Novo Nordisk A/S, Novo Nordisk Park, 2730 Måløv (Denmark); Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvalsensvej 40, 1870 Frederiksberg (Denmark).
authors.forename: Ebbe
authors.initials: E
authors.lastname: Engholm
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Hansen
authors.forename: Eva
authors.initials: E
authors.lastname: Johansson
authors.forename: Holger M
authors.initials: HM
authors.lastname: Strauss
authors.forename: Tine N
authors.initials: TN
authors.lastname: Vinther
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.forename: Frantisek
authors.initials: F
authors.lastname: Hubálek
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Kjeldsen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D015870:Gene Expression; D006168:Guinea Pigs; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017526:Receptor, IGF Type 1 / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D007328:Insulin; D017526:Receptor, IGF Type 1; D011972:Receptor, Insulin
keywords: guinea pig insulin; insulin expression; metal affinity; protein expression; receptors