Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification

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 feature...

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Published in:Protein expression and purification 2016-03, Vol.119, p.102-109
Main Authors: Zieliński, Marcin, Wójtowicz-Krawiec, Anna, Mikiewicz, Diana, Kęsik-Brodacka, Małgorzata, Cecuda-Adamczewska, Violetta, Marciniak-Rusek, Alina, Sokołowska, Iwona, Łukasiewicz, Natalia, Gurba, Lidia, Odrowąż-Sypniewski, Michał, Baran, Piotr, Płucienniczak, Grażyna, Płucienniczak, Andrzej, Borowicz, Piotr, Szewczyk, Bogusław
Format: Article
Language:English
Subjects:
Amidine-Lyases - biosynthesis
Amidine-Lyases - chemistry
Amidine-Lyases - isolation & purification
Animals
Blood Glucose
Chinese hamster ovary cell
CHO Cells
Chromatography, Gel
Chromatography, High Pressure Liquid
Cricetinae
Cricetulus
Diabetes Mellitus, Experimental - drug therapy
Dihydrofolate reductase
Drug Evaluation, Preclinical
Female
Human peptidylglycine α-amidating monooxygenase
Hypoglycemic activity
Hypoglycemic Agents - chemistry
Hypoglycemic Agents - pharmacology
Insulin - analogs & derivatives
Insulin - chemistry
Insulin - pharmacology
Insulin amide
Male
Methotrexate
Mixed Function Oxygenases - biosynthesis
Mixed Function Oxygenases - chemistry
Mixed Function Oxygenases - isolation & purification
Rats, Wistar
Recombinant Proteins - biosynthesis
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
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Summary: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 
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2015.11.017