Optimization Research On Ribonuclease Onconase Preparation

Leopard frog enzyme (Onconase, referred to as the ONC) is from the arctic leopard frog oocyte and early embryo extract RNA enzymes, belong to RNase A super family, study confirms its for many cancer cells and solid tumors have damage effect.With a strain express leopard frog enzyme gene engineering bacteria as the research object, respectively in shake flask and3.7L small fermentation tank of engineering bacteria fermentation process has been optimized, and determined the inoculation quantity2%, IPTG concentration is0.5mM, induction time3.5hours, bacteria body leopard frog enzyme expression quantity of intracellular protein over30%of the total.Further to leopard frog enzyme purification process to explore, the establishment of a somatic crushing, inclusion body wash, protein renaturation, protein purification process. In this paper the renaturation method is mainly to dilution method. In order to get the optimal renaturation conditions, the experiment on different renaturation temperature research, the results show that as the renaturation temperature at4℃, renaturation effect optimal; For renaturation buffer of salt (mainly arginine and urea choice) study, the results show that as the renaturation buffer to add0.5M of arginine, renaturation effect optimal; For renaturation buffer of urea concentration, the results showed that when the renaturation buffer to add urea concentration of2M, renaturation effect optimal; GSSG to join renaturation buffer time, the results showed that in2M urea system, inclusion body join buffer before join GSSG renaturation effect optimal. Four kinds of protein renaturation system research (including urea concentration of2M,1.5M urea concentration, including arginine, acid sinking method used), and the results show that each renaturation system can make leopard frog enzyme electrophoresis purity above90%,the protein recovery rate above30%in arginine system.