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| Cloning, Expression, Purification and Its Biological Activity Study of a Novel GIP Analog |
| SONG Wen-cheng1,JIN Ming-fei1,2,LI Dong-qing1,ZHAO Li-fen1,ZHANG Hong-dan1,HUANG Jing1,WU Zi-rong1 |
1.School of Life Science -East China Normal University, Shanghai 200062,China
2.Institute Pasteur of Shanghai Chinese Academy of Sciences, Shanghai 200025,China |
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Abstract Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. It’s difficult to be applied because of its extremely short half life time. Modifying the dipeptidase IV recognition amino acid can prolong its half life time. Different GIP mutant genes that were obtained by artificial synthesizing or site-directed mutagenesis were cloned into the vector pET32a(+). These plasmids were expressed in Escherichia coli. The 6-histamine fused proteins were purified by NTA-Ni2+ affinity chromatography, respectively. Purified fusion proteins were then digested with enterokinase and purified with another affinity chromatography and desalting. The biological activities of GIP analogs were tested in vivo in Kuming mice or diabetic db/db mice at a dose of 48nmol/kg by intraperitoneal injection associated with injection of glucose at 18mmol/kg. The results showed that the half life time of analog mGIP243 in Kunming mice was extended to 90 minutes (p<0.001), which was much longer than those of ala to ser analog mGIP2 (30 minutes) or native GIP(shorter than 30 minutes). The oral glucose tolerance tests in Kuming mice and db/db mice both supported the results with a magnificent glucose lowering effects (p<0.01 VS saline group) by the end of a 60 minutes test. Interestingly, the novel GIP analog mGIP243 played good glucose lowering effect in diabetic mice. In conclusion, the novel GIP analog mGIP243 is a good drug candidate for diabetes.
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Received: 13 January 2010
Published: 12 June 2010
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Cite this article:
SONG Wen-Cheng, JIN Meng-Fei, LI Dong-Jing, DIAO Li-Fen, ZHANG Hong-Dan, HUANG Jing, TUN Zi-Rong. Cloning, Expression, Purification and Its Biological Activity Study of a Novel GIP Analog. China Biotechnology, 2010, 30(06): 38-43.
URL:
https://manu60.magtech.com.cn/biotech/Q789 OR https://manu60.magtech.com.cn/biotech/Y2010/V30/I06/38
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| ［1］ Buchan M T,Polak J M, Capella C, et al. Electronimmunocytochemical evidence for the Kcell localization of gastric inhibitory polypeptide (GIP) in man. Histochemistry, 1978, 56(1): 3744.
［2］ Brown J C, Dryburgh J R, Ross S A, et al. Identification and actions of gastric inhibitory polypeptide. Recent Prog Horm Res, 1975, 31: 487532.
［3］ Holst J J. Gastric inhibitory polypeptide analogues: do they have a therapeutic role in diabetes mellitus similar to that of glucagonlike peptide1? BioDrugs, 2002, 16(3): 175181.
［4］ Gault V A, O’Harte F P M, Flatt P R. Glucosedependent insulinotropic polypeptide (GIP): antidiabetic and antiobesity potential? Neuropeptides, 2003, 37(5): 253263.
［5］ Meier J J, Nauck M A, Schmidt W E, et al. Gastric inhibitory polypeptide: the neglected incretin revisited. Regulatory Peptides, 2002, 107(13): 113.
［6］ 马义,李弘剑,周天. 基因重组胰高血糖素样肽21衍生多肽的表达和产物纯化与鉴定. 中国生物工程杂志, 2009, 29(6): 16. Ma Y, Li H J, Zhou T. China Biotechnology, 2009, 29(6): 16.
［7］ Dupre J, Ross S A, Watson D, et al. Stimulation of insulin secretion by gastric inhibitory polypeptide in man. J Clin Endocrinol Metab, 1973, 37(5): 826828.
［8］ Nauck M A, Heimesaat M M, ?rskov C, et al. Preserved incretin activity of glucagonlike peptide 1 ［7– 36 amide］ but not of synthetic human gastric inhibitory polypeptide in patients with type2 diabetes mellitus. J Clin Invest, 1993, 91(1): 301307.
［9］ May J M, Williams R H. The effect of endogenous gastric inhibitory polypeptide on glucoseinduced insulin secretion in mild diabetes. Diabetes, 1978, 27(8): 849855.
［10］ Kindmark H, Pigon J, Efendic S. Glucosedependent insulinotropic hormone potentiates the hypoglycemic effect of glibenclamide in healthy volunteers: evidence for an effect on insulin extraction. J Clin Endocrinol Metab, 2001, 72(5): 20152019.
［11］ O’Harte F PM, Mooney M H, Flatt PR. NH2terminally modified gastric inhibitory polypeptide exhibits aminopeptidase resistance and enhanced antihyperglycemic activity. Diabetes, 1999, 48(4): 758765.
［12］ O’Harte F PM, Mooney M H, Kelly C M, et al. Improved glycaemic control in obese diabetic ob/ob mice using Nterminally modified gastric inhibitory polypeptide. J Endocrinol, 2000, 165(3): 639648.
［13］ Hinke S A, Manhart S, KühnWache K, et al. ［Ser2］ and ［Ser(P)2］incretin analogs. J Biol Chem, 2004, 279(6): 39984006.
［14］ 汪洋,马明浩,冯震，等.重组人谷氨酸脱羧酶65的表达及活性研究.中国生物工程杂志, 2009, 29(4): 1216. Wang Y, Ma M H, Feng Z, et al. China Biotechnology, 2009, 29(4): 1216.
［15］ Sharma K, McCue P and Dunn1S R. Diabetic kidney disease in the db/db mouse.Am J Physiol Renal Physiol, 2003,284(6): F1138F1144.
［16］ 金明飞,徐伟东,黄静,等.基因串联原核高效表达胸腺肽α1.中国生物工程杂志, 2007, 27(1):1115. Jin M F, Xu W D, Huang J, et al. China Biotechnology, 2007, 27(1):1115.
［17］ 尤晓娟,刘劲松.重组人SNX9蛋白在大肠杆菌中的表达、纯化及性质分析.中国生物工程杂志,2008, 28(8): 1117. You X J, Liu J S. China Biotechnology, 2008, 28(8): 1117. |
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