Expression, purification, and biological activity of the recombinant pramlintide precursor
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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.
KeywordsAmylin Pramlintide precursor Amidation L6 myotubes Gastric emptying
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.
- Butt TR, Edavettal SC, Hall JP, Mattern MR (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43:1–9Google Scholar
- Eipper BA, Richard E (1988) Peptide alpha-amidation. Annu Rev Physiol 50:333–344Google Scholar
- Eipper BA, Milgram SL, Husten EJ, Yun HY, Mains RE (1993) Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains. Protein Sci 2:489–497Google Scholar
- Jung S, Mysliwy J, Spudy B, Lorenzen I, Reiss K, Gelhaus C, Podschun R, Leippe M, Grötzinger J (2011) Human beta-defensin 2 and beta-defensin 3 chimeric peptides reveal the structural basis of the pathogen specificity of their parent molecules. Antimicrob Agents Chemother 55:954–960Google Scholar
- Kim KH, Seong BL (2001) Peptide amidation: production of peptide hormones in vivo and in vitro. Biotechnol Bioprocess Eng 6:244–251Google Scholar
- Leighton B, Cooper GJ (1988) Pancreatic amylin and calcitonin gene-related peptide cause resistance to insulin in skeletal muscle in vitro. Nature 335:632–635Google Scholar
- Nyholm B, Orskov L, Hove KY, Gravholt CH, Møller N, Alberti KG, Moyses C, Kolterman O, Schmitz O (1999) The amylin analog pramlintide improves glycemic control and reduces postprandial glucagon concentrations in patients with type 1 diabetes mellitus. Metabolism 48:935–941PubMedCrossRefGoogle Scholar
- Ray MV, Van Duyne P, Bertelsen AH, Jackson-Matthews DE, Sturmer AM, Merkler DJ, Consalvo AP, Young SD, Gilligan JP, Shields PP (1993) Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide. Biotechnology (N Y) 11:64–70CrossRefGoogle Scholar