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
Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
Institute of Biotechnology and Antibiotics, Starościńska 5, Warszawa 02-516, Poland.
Protein Expr Purif. 2016 Mar;119:102-9. doi: 10.1016/j.pep.2015.11.017. Epub 2015 Nov 22.
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.
具有催化活性的肽基甘氨酸α-酰胺化单加氧酶（PAM）的可得性应为药理学研究中检验其在生物活性肽化学酶促合成中的潜在用途提供手段。降血糖活性是胰岛素衍生物最重要的特征之一。发现甘精胰岛素酰胺显示出一种时间/效应曲线，该曲线明显更平缓，因此比甘精胰岛素本身更具优势。本研究的目的是获得重组PAM并将其用于胰岛素类似物的酰胺化。我们在培养的CHO dhfr-细胞中稳定表达了重组PAM。重组PAM通过分级硫酸铵沉淀和离子交换色谱进行部分纯化。该酶用于修饰甘氨酸延伸的A22(G)-B31(K)-B32(R)人胰岛素类似物（GKR）。通过HPLC和质谱分析α-酰胺化胰岛素。比较了酰胺化和未酰胺化胰岛素的降血糖活性。药效学效应基于对链脲佐菌素诱导的高血糖Wistar大鼠的血糖浓度测量。在2.5-7.5 U/kg体重的范围内皮下单次给药后，评估了长达36小时的总体血糖曲线。大鼠实验证实，与接受0.9% NaCl的对照组相比，GKR-NH2具有统计学意义（P < 0.05）的降血糖活性。GKR-NH2曲线的特征是起效相当快（0.5-2.0小时）且恢复到初始值的时间相当长。GKR-NH2是糖尿病护理中降血糖药物产品的候选物。此外，这项工作还为制备任何其他具有C端酰胺化的重组生物活性肽提供了一种有价值的替代方法。
