title: Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification.
abstract: The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland. Electronic address: marcinzielinski2@wp.pl.
authors.forename: Marcin
authors.initials: M
authors.lastname: Zieliński
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Wójtowicz-Krawiec
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Diana
authors.initials: D
authors.lastname: Mikiewicz
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Małgorzata
authors.initials: M
authors.lastname: Kęsik-Brodacka
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Violetta
authors.initials: V
authors.lastname: Cecuda-Adamczewska
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Alina
authors.initials: A
authors.lastname: Marciniak-Rusek
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Iwona
authors.initials: I
authors.lastname: Sokołowska
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Natalia
authors.initials: N
authors.lastname: Łukasiewicz
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Lidia
authors.initials: L
authors.lastname: Gurba
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Odrowąż-Sypniewski
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Baran
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Grażyna
authors.initials: G
authors.lastname: Płucienniczak
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Andrzej
authors.initials: A
authors.lastname: Płucienniczak
authors.affiliation: Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Borowicz
authors.affiliation: Intercollegiate Faculty of Biotechnology of the University of Gdańsk and the Medical University of Gdańsk, Kładki 24, Gdańsk 80-822, Poland.
authors.forename: Bogusław
authors.initials: B
authors.lastname: Szewczyk
mesh_terms: D019760:Amidine-Lyases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D001786:Blood Glucose; D016466:CHO Cells; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy; D004353:Drug Evaluation, Preclinical; D005260:Female; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000494:pharmacology; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000494:pharmacology; D008297:Male; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001786:Blood Glucose; D007004:Hypoglycemic Agents; D007328:Insulin; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C544787:PAM protein, human; D019760:Amidine-Lyases
keywords: Chinese hamster ovary cell; Dihydrofolate reductase; Human peptidylglycine α-amidating monooxygenase; Hypoglycemic activity; Insulin amide; Methotrexate