Expression, purification, and characterization of C-terminal amidated glucagon in Streptomyces lividans.
Glucagon, a peptide hormone produced by alpha-cells of Langerhans islets, is a physiological antagonist of insulin and stimulator of its secretion. In order to improve its bioactivity, we modified its structure at the C-terminus by amidation catalyzed by a recombinant amidase in bacterial cells. The human gene coding for glucagon-gly was PCR amplified using three overlapping primers and cloned together with a rat alpha-amidase gene in plasmid pMGA. Both genes were expressed under control of the strong constitutive promoter of aph and secretion signal melC1 in Streptomyces lividans. With Phenyl-Sepharose 6 FF, Q-Sepharose FF, SP-Sepharose FF chromatographies and HPLC, the peptide was purified to about 93.4% purity. The molecular mass of the peptide is 3.494 kDa as analyzed by MALDI TOF, which agrees with the theoretical mass value of the C-terminal amidated glucagon. The N-terminal sequence of the peptide was also determined, confirming its identity with human glucagon at the N-terminal part. ELISA showed that the purified peptide amide is bioactive in reacting with glucagon antibodies.
Qi
Xiaoqiang
X
Ministry of Health, Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Tiantan, Beijing, China.
Jiang
Rong
R
Yao
Cheng
C
Zhang
Ren
R
Li
Yuan
Y
D000581:Amidohydrolases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005934:Glucagon / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008970:Molecular Weight; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D048170:Streptomyces lividans / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D005934:Glucagon; D000581:Amidohydrolases; C019665:amidase