Recombinant Tissue-type Plasminogen Activator Variants (reteplase) Refolding

Cardiovascular diseases are the main diseases which threaten to the lives of human ,among then are acute myocardial infarction, pulmonary embolism, brain infarction, deep venous thrombosis, outer aortic infarction. According to estimate of WHO, approximately .twelve million people die of cardiovascular diseases each year in the world. Therefore, it is an urgent requirement for design and produce some kinds of medicines to clear away thrombus.Reteplase ,one of the most effective medicines for Cardiovascular diseases ,has many advantages,such as easily produce Jong half life , effective for clear away thrombus .little side effect and etc.The gene of reteplase is subcloned into the pet22b and the expression vector was transformed into E.coli BL21(DE3).The protein expresses as inclusion body, which has the feature of high expression level ,resisting hydrolyzation of enzyme, no toxicity to host. If the protein can be easily refolded , Fa certain , inclusion body is a appropriate choiceWe studied the expression condition of reteplase and got an optimized condition for increasing the expression level of inclusion body. culture medium : Tryptone 10 g/L.Yeast reextract powder 5 g/L, NaCl 10 g/L, Na₂HPO₄ 12H₂O 6 g/L, KH₂PO₄ 3 g/L,MgSO₄ 7H₂O 1 g/L,Glucose 5 g/L,PH=7.2 ,37 ℃., oxygen dissolve was kept 100% at the beginning 3 hour of culture and the following process was controlled to 40%.350ml 200 g/L Tryptone was added conti nuously after 3 h of fermentation. When OD₆₀₀ get to 20 , IPTG is added to final concentration 80μM , glucose concentration is kept under 0.1 g/L, 40 ℃,then harvest bacteria after 4h induced by IFTG .Using this protocol, we have been able to obtain 3g/L inclusion bodies.In order to explore the efficient methods of protein refolding in vitro, we attempted to use the following methods.