Protein Expression, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark.
J Pept Sci. 2010 Sep;16(9):473-9. doi: 10.1002/psc.1264.
Backbone cyclic insulin was designed and prepared by reverse proteolysis in partial organic solvent of a single-chain precursor expressed in yeast. The precursor contains two loops to bridge the two chains of native insulin. The cyclisation method uses Achromobacter lyticus protease and should be generally applicable to proteins with C-terminal lysine and proximal N-terminal. The presence of the ring-closing bond and the native insulin disulfide patterns were documented by LC-MS peptide maps. The cyclic insulin was shown to be inert towards degradation by CPY, but was somewhat labile towards chymotrypsin. Intravenous administration of the cyclic insulin to Wistar rats showed the compounds to be equipotent to HI despite much lower insulin receptor affinity.
通过在酵母中表达的单链前体的部分有机溶剂中的反向蛋白水解，设计并制备了骨干环胰岛素。该前体包含两个环，用于桥接天然胰岛素的两条链。环化方法使用溶壁微球菌蛋白酶，并且应该普遍适用于具有 C 末端赖氨酸和近端 N 末端的蛋白质。通过 LC-MS 肽图证明了闭环键和天然胰岛素二硫键模式的存在。尽管胰岛素受体亲和力低得多，但环状胰岛素对 CPY 的降解表现出惰性，但对糜蛋白酶的稳定性稍差。将环状胰岛素静脉内给予 Wistar 大鼠，尽管其胰岛素受体亲和力低得多，但化合物的效价与 HI 相当。
