Cloning And Expression Of Rat Proinsulin Coding Sequence

Diabetes is a common disease which is caused by genetic and enviorment factors, but in the study process of the pathogenesis, we use human insulin to take place of the animal insulin, which makes many problems.So cloning and expression of rat insulin is very important.We clone the rat proinsulin coding sequence and expressing in the E.coli.We extract the total RNA from the rat pancreas and reverse transcribed to cDNA, perform polymerase chain reaction (PCR) to clone the exons of rat insulin coding gene, and then clone it to pGEM-T Easy. After confirmatory sequencing, we insert the aimed gene to pQE-30Xa, which is prokaryotic expression vector, and then transform it respectively to E.coli M15 [pREP4] to express aimed protein. Perform SDS-PAGE to check the molecular weight and quantity of proinsulin. Operate Western-blotting to further identify. Optimize the expressing condition to express pQE-30Xa/proinsulin in E.coli M15 [pREP4] in batches. Proinsulin is 19mg in per liter culture medium. The recombinant proinsulin is 28.24% in total germ protein. The recombinant proinsulin has 6 X His purification tag, so it can be seperated with Ni2+-IDA resin affinity chromatography via the bond between histidine and Ni2+. After rinsing the mixed other kinds of protein, we can wash the aimed protein from resin. By Ni2+-IDA resin affinity chromatography we can purify the aimed protein produced in batches.We obtain the rat insulin by the enzyme cutting and purify it by the Ni2+-IDA resin again. The final products showed one band by Tricine-SDS-PAGE analysis and were identical with the native rat insulin by ELISA.We obtain 26g wet E.coli from 1 liter culture media,respectively, by the E.coli high-density fermentation.