Studies On Denaturation, Renaturation And Purification Of Reteplase

Reteplase (Recombinant tissue plasminogen activator variant)represents one of the third generation thrombolytic drugs. Reteplase manifests the advantages in both manufactory and clinic: large scale production, prolonged half-life time, thrombolytic potency and fev/er side effects. Reteplase is the non-glycosylated, truncated mutant of wild-type t-PA molecule. It is a domain-delete protein variant, which only contains kringle II, protease domain and N-terminus three amino acids. The protein sequence contained 355 amino acids and 9 disulfide bonds with a calculated MW of 39,000 Daltons.The bacterium of E. coli had been choosen to produce the protein. Recombinant proteins can be expressed at high level. In many cases, the accumulation of the protein product in inactive insoluble deposits inside the cells, called inclusion bodies, often occurs. It is a skillful technology to produce inclusion bodies by high-density fermentation, and the induction of IPTG is very crucial. The optimization of induction can cause the high expression levels of inclusion bodies and avoid contamination of IPTG with high concentration. Under the induction of IPTG, the recombinant protein can aggregate in the cell and develop inactive inclusion bodies, which contain some nucleic acids and other proteins such as membraned proteins in addition to recombinant proteins. Therefore, purification of inclusion bodies is very useful to get the purified and actived Reterplase. Low-concentration denaturants such as urea can clear away some impurities, but not thoroughly. However, Efficient purification can be reached by the three-step method : salt washing- urea washing-water washing.The purpose of denaturation is the dissolution of inclusion bodies, then inactive Reteplase can become active so that it can improve the renaturation ratio and reduce the production cost. Appropriate denaturants are necessary to...