Preliminary Studies On Construction, Expression And Renaturation Of A Single-Chain Recombinant Human Insulin Analogue (HI-270)

For many years, Diabetes Mellitus (DM) and its complications (such as Ketoacid uremia, stroke and blindness caused by macroangiopathy and microvascular impairment, etc.), have together seriously cmpromised human health and quality of lives, it has became one of three causes which result in death with cardiovascular disease and cancer. Currently, in China there are 920 million diabetes patients, and 1480 million pre-diabetes, ranking first place globally. Insulin at present is the best and safetest drug for Diabetes. With DM rate daily up, the need of insulin market is increasing; therefore, insulin shows great future both in biopharmacy and in research fields.Since 1922 when insulin was first extracted from dog pancreas in 1921 followed by immediate application in clinical treatment for DM, and the historical recombinant insulin from gene engineering, the application and development of insulin has undergone a long path. Mature insulin consists of A-chain (21 amino acids) and B-chain (30 amino acids), and its molecule size is about 5.7 KD. The A-chain and B-chain are connected by an intra-A disulfide bridge (A6-A11) and two inter-disulfide bridges (A7-B7, A20-B19). Following by the development of molecular biology, protein and its related technologies, more and more researches are focused on how to highly effectively, simply and cheaply manufacture recombinant insulin with novel action profiles. This study aims to increase the expression output of recombinant insulin, by designing new construction plasmid, choosing and optimal expression system and the best ferment condition building and so on, at the same time, building real and practical renaturation methods, to create conditions for late downstream purification, trypsin and apart.In this experiment, we used plasmid vector pET-39b, E.coli BL21 as expression system, and designed a construct with novel signal peptide and a six histeins peptide, which increases expression output and simplifying purification by Ni-resin; A-chain and B-chain independently connected a certain minical connecting peptide by two arginines, not only no effecting on refolding and renaturation of recombinant protein, but also good for late renaturation method, to create conditioyns for late downstream purification, trypsin and apart, to achieve two chains recombinant insulin. We had the following yield: E.coli 840 mg/L (wet weight), inclusion bodies 367.89 mg/L (wet weight), corresponding to an expression level of 43.8%, object protein, recombinant insulin 25% in total proteins. After reduction and renaturation, we recovered single chain human insulin analogue H-270T. Technologies such as Tricine-SDS-PAGE, HPLC and CD will be applied as the next step to validate correction of object protein.