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Applied Microbiology and Biotechnology

, Volume 98, Issue 18, pp 7837–7844 | Cite as

Expression, purification, and biological activity of the recombinant pramlintide precursor

  • Hao Hu
  • Qi Xiang
  • Hui Liu
  • Hongyan Qu
  • Xin Tang
  • Xue Xiao
  • Qihao Zhang
  • Zhijian Su
  • Yadong Huang
Biotechnologically relevant enzymes and proteins

Abstract

Pramlintide is an artificially designed protein which has the same function as amylin in human body. This protein is extremely difficult to synthesize through prokaryotic expression method because of its two essential active sites, intrachain disulfide bond and C-terminal amide group. Since α-amidating monooxygenase is widely distributed in human and animal, it is possible to use pramlintide precursor with an additional C-terminal glycine (PAG), which is the potential substrate of α-amidating monooxygenase, for in vivo applications. The recombinant PAG was expressed in Escherichia coli using the small ubiquitin-related modifier (SUMO) as the molecular chaperone, and the optimal fusion expression level reached to 36.3 % of the total supernatant protein. Under optimal conditions in a 10-L fermentor, the recombinant PAG was obtained with a purity of greater than 95 %, and the average expression level was reached to 20 mg/L. The authenticity and the intrachain disulfide bridge of PAG were confirmed by Western blotting and matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS) as well as N-terminal sequencing of protein. Based on an L6 myoblast cell model in vitro and an animal model of gastric emptying in vivo, the results of activity revealed that PAG showed a lower biological activity in vitro but has almost the same activity as the chemically synthesized pramlintide in vivo.

Keywords

Amylin Pramlintide precursor Amidation L6 myotubes Gastric emptying 

Notes

Acknowledgments

This work was supported by the National Key New Drug Creation of China (NOs. 2012ZX09103301-034 and 2011ZX09401-307), the Guangdong Province Ministry of Education that produces study that grinds the union project of China (2009B090300091), the Science and Technology Major Project of Guangdong Province (2011A080502014, 2012A080201010), and the Guangzhou Municipal Technical Innovation Fund for Medium and Small-Size Enterprise of China (2010Q-p012).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2014_5699_MOESM1_ESM.pdf (209 kb)
ESM 1 (PDF 209 KB)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Hao Hu
    • 1
  • Qi Xiang
    • 3
  • Hui Liu
    • 2
    • 3
  • Hongyan Qu
    • 1
  • Xin Tang
    • 1
  • Xue Xiao
    • 2
    • 3
  • Qihao Zhang
    • 2
    • 3
  • Zhijian Su
    • 3
  • Yadong Huang
    • 1
    • 2
  1. 1.National Engineering Research Center of Genetic MedicineJinan UniversityGuangzhouChina
  2. 2.Institute of BiomedicineJinan UniversityGuangzhouChina
  3. 3.Guangdong Provincial Key Laboratory of Bioengineering MedicineJinan UniversityGuangzhouChina

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