Expression and purification of recombinant human insulin from E. coli 20 strain

Abstract

The number of people with diabetes is estimated to be over 370 million, in 2030 it will increase to 552 million. In Poland, the number of people with diabetes is estimated to be 3.5 million (9.1%). According to the estimates of the International Diabetes Federation, the percentage of patients in the adult Polish population will increase to around 11% over the next 20 years. Despite the appearance of insulin analogues on the pharmaceutical market, insulin delivery is still the most effective method of pharmacotherapy in cases of extremely high hyperglycemia. A new bacterial host strain (Escherichia coli 20) was obtained at the Institute of Biotechnology and Antibiotics and a new pIBAINS expression vector was constructed that provides greater efficiency in the production of recombinant human insulin. In the IBA Bioengineering Department, successful attempts were made to produce recombinant human insulin on a laboratory and quarter-technical scale, and several batches were performed on a semi-technical scale. The production process has been divided into several stages: 1. biosynthesis of insulin in the fermenter, 2. isolation, purification and dissolution of inclusion bodies, 3. protein renaturation, 4. enzymatic reaction with trypsin, 5. multi-stage purification of insulin using low-pressure and HPLC techniques. At each stage of insulin production, qualitative and quantitative analyses were performed to confirm identity and purity. In particular, the molecular weight of insulin, the amount of insulin and the content of protein impurities were studied. The results of these experiments are presented in this work.