Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression, Purification, and Characterization of C-Terminal Amidated Glucagon in Streptomyces lividans

  • Qi, Xiaoqiang (Ministry of Health Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College) ;
  • Jiang, Rong (Ministry of Health Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College) ;
  • Yao, Cheng (Ministry of Health Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College) ;
  • Zhang, Ren (Ministry of Health Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College) ;
  • Li, Yuan (Ministry of Health Key Laboratory of Biotechnology of Antibiotics, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College)
  • Published : 2008.06.30
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Abstract

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.

Keywords

References

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