title: Study on preparation and unique properties of a novel insulin analogue with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain.
abstract: A novel insulin analog, PIns, with N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at human regular insulin B-chain was acquired through gene engineering. Preproinsulin for PIns was cloned and expressed using a bacterial expression system at a high level (72.1%) as fusion protein carrying a modified thioredoxin N-terminal region (1-21) linked to N-terminus of proinsulin by a lysine residue. Purified fusion protein was refolded and converted into PIns by a single enzymatic reaction. After PIns was purified, the homogeneity of it was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectronic focusing electrophoresis, amino acid composition analysis and mass spectrometry methods. A decreased tendency of self-association of PIns as compared with regular insulin was demonstrated by the size exclusion HPLC analysis. When subcutaneously administrated into normal rats, the PIns showed a faster rate of onset of action and a shorter duration of action compared with regular insulin, similar to the pharmacokinetic characteristics of insulin Lispro. These results showed that PIns is a rapid insulin analog. Furthermore, the N-terminal Arg-4, Pro-3, Lys-2, Pro-1extension at insulin B-chain can be excised by DPPIV and recombinant peptidase with DPPIV-like activities. It is suggested that PIns serves as an artificial insulin precursor and can be transformed to regular insulin in vivo due to the truncation of N-terminal sequence of PIns B-chain by DPPIV.
authors.affiliation: Minigene Pharmacy Laboratory, Biopharmaceutical College, China Pharmaceutical University, Nanjing 210009, China. minigene2@yahoo.com.cn
authors.forename: Lu
authors.initials: L
authors.lastname: Yong
authors.forename: Zhang
authors.initials: Z
authors.lastname: Huiyong
authors.forename: Fang
authors.initials: F
authors.lastname: Jing
authors.forename: Wang
authors.initials: W
authors.lastname: Huaqian
authors.forename: You
authors.initials: Y
authors.lastname: Kejun
authors.forename: Yang
authors.initials: Y
authors.lastname: Yanan
authors.forename: Liu
authors.initials: L
authors.lastname: Suli
authors.forename: Wang
authors.initials: W
authors.lastname: Xuejun
authors.forename: Meng
authors.initials: M
authors.lastname: Yuhan
authors.forename: Cao
authors.initials: C
authors.lastname: Rongyue
authors.forename: Fan
authors.initials: F
authors.lastname: Hao
authors.forename: Li
authors.initials: L
authors.lastname: Taiming
authors.forename: Liu
authors.initials: L
authors.lastname: Jingjing
mesh_terms: D000818:Animals; D005818:Genetic Engineering; D006801:Humans; D007279:Injections, Subcutaneous; D007328:Insulin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000737:chemistry / Q000493:pharmacokinetics*; D008297:Male; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: D007328:Insulin; C546774:PIns peptide; D011993:Recombinant Fusion Proteins