title: Soluble insulin analogs combining rapid- and long-acting hypoglycemic properties - From an efficient E. coli expression system to a pharmaceutical formulation.
abstract: The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Diana
authors.initials: D
authors.lastname: Mikiewicz
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Bierczyńska-Krzysik
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Sobolewska
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Stadnik
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Bogiel
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Pawłowska
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Wójtowicz-Krawiec
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Piotr A
authors.initials: PA
authors.lastname: Baran
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Natalia
authors.initials: N
authors.lastname: Łukasiewicz
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Romanik-Chruścielewska
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Sokołowska
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Jacek
authors.initials: J
authors.lastname: Stadnik
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Borowicz
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Grażyna
authors.initials: G
authors.lastname: Płucienniczak
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, 02-516 Warsaw, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Płucienniczak
mesh_terms: D004926:Escherichia coli / Q000235:genetics*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000008:administration & dosage* / Q000031:analogs & derivatives*; D004364:Pharmaceutical Preparations / Q000008:administration & dosage; D012995:Solubility
chemical_list: D007004:Hypoglycemic Agents; D007328:Insulin; D004364:Pharmaceutical Preparations