The Recombinant Expression And Purification Of Monomeric B27 Lys Destripeptide Insulin Precursor-MIP

Diabetes is a kind of metabolic diseases characterized by hyperglycemia. Without timely treatment, it will trigger a series of complications. Now, it has become one of the greatest killers threatening the human health. Insulin is a kind of peptide hormone secreted by pancreatic beta cells. It has been one of the most efficient pharmaceuticals against diabetics in clinical applications. So the study of insulin is extremely important.Insulin has a history of 90 years since its discovery. During the last 90 years, according to the source and chemical structure of different insulin, the insulin products experienced three stages from animal insulin, human insulin and insulin analogue. In the 1990 s, via modifying the amino acid sequence and structure of insulin with genetic engineering, the scientist synthesized the insulin analogs. Insulin analogs changed the pharmacokinetic characteristics of insulin which opened a new chapter in medical insulin history. The fast-acting insulin analogs is obtained via genetic engineering which display faster absorption kinetics compared with human insulin, therefore, more closely mimic endogenous insulin secretion.In recent years, because of its fast absorption and high safety, the fast-acting insulin analogs have attracted more and more attention. Now, there are three commercially-available fast-acting insulin analogs(insulin lispro [ILis; HumaLogR, EliLilly], insulin aspart [IAsp; NovoRapidR, Novo Nordisk], and insulin glulisine [IGlu; ApidraTM, Sanofi-Aventis]).B27K-DTrI insulin(human insulin with B28-30 removed and B27 Thr replaced by Lys) is a kind of fast-acting insulin anologs which successfully prepared in pichia pastoris expression system. The B27K-DTrI insulin was reported to have superior monomeric property with 80% insulin activity in vivo according to mice convulsion method. The size exclusion chromatography showed that the recombinant B27K-DTrI insulinhas better monomer properties than Molecular sieve experiment showed that recombinant B27K- DTrI monomer properties is better than lai monomer properties of insulin lispro. So, it has potential use as a new fast-acting insulin anolog. But no matter using Pichia pastoris expression system or IMPACT-TWIN system, the solubility of B27K-DTrI is low. The subsequent operations were complex, and the yield was too low.MIP is the precursor of B27K-DTrI, which can be prepared by trypsin hydrolysis. So, obtaining high yield and high purity of MIP is the key to get final product B27K-DTrI. Here, we reported the preparation of MIP from E. coli by ppSUMO expression system. The solubility of precursor fusion protein was improved using the system. The purification process was simplified without tedious renaturation operations. It can improve the yield of SUMO-MIP fusion protein. The one-step affinity purification produced about 30.1 mg target fusion protein of >75 % purity from per liter medium of TB. By ion exchange chromatography and size chromatography, we got 17.5 mg target fusion protein of >95 % purity from per liter medium. Finally the SUMO-MIP fusion protein was converted to MIP by ULP1 hydrolysis. It laid the foundation for the preparation of B27K-DTrI.