title: Expression, purification, and biological activity of the recombinant pramlintide precursor.
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.
authors.affiliation: National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, 510632, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Hu
authors.forename: Qi
authors.initials: Q
authors.lastname: Xiang
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Hongyan
authors.initials: H
authors.lastname: Qu
authors.forename: Xin
authors.initials: X
authors.lastname: Tang
authors.forename: Xue
authors.initials: X
authors.lastname: Xiao
authors.forename: Qihao
authors.initials: Q
authors.lastname: Zhang
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Su
authors.forename: Yadong
authors.initials: Y
authors.lastname: Huang
mesh_terms: D000818:Animals; D002460:Cell Line; D004926:Escherichia coli / Q000378:metabolism*; D005746:Gastric Emptying / Q000187:drug effects; D058228:Islet Amyloid Polypeptide / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D058228:Islet Amyloid Polypeptide; D011993:Recombinant Fusion Proteins; C105254:pramlintide