Research On The Expression,Purification And Directed Evolution Of Antifreeze Protein Of Tenebrio Molitor

Antifreeze proteins(AFPs)belong to a family of proteins that have the common ability to protect organisms from damage at freezing or sub-freezing conditions(closed to freezing temperature and above).AFPs can inhibit the growth of ice crystal and lower the freezing point non-colligatively while leaving the melting point unchanged(the temperature gap is called thermal hysteresis),which would help organisms survive at low temperature.Althrough AFPs have been found in a wide variety of organisms such as fishes,insects,plants,microalgae and even bacteria living in Arctic and Antarctic regions,the problem is that the absolute amount is very small.The insect antifreeze proteins are hyperactive in antifreeze proteins and have specific activities 10-100 times greater than those of fish AFPs.Therefore,insect antifreeze proteins have raised great interests among researchers due to their attractive characteristics and potentially great commercial value.Both scientific research and commercial application require a large number of soluble AFPs.In this paper,yellow mealworm antifreeze protein(TmAFP)as the target protein was successfully expressed in Escherichia coli by means of genetic engineering and obtained with biology activity through the purification by Ni-NTA column,which provided strong and valuable technical support for scientific research and commercial application.The main findings are as follows:(1)The expression plasmid pET-D sbA-TmAFP was successfully constructed with the original vector pET-DsbA,which contains the ampicillin resistance gene.(2)Optimization of the condition of antifreeze protein expressed in E.coli,such as bacteria host,OD600,IPTG concentration and the induction temperature,etc.Finally the best expression conditions was known:the E.coi Shuffle T7 with plasmid pET-DsbA-TmAFP was incubated at 30? until OD600 reached 0.6,the final concentration of 1 mM IPTG was added and the culture was transferred to a 18? shaker for 16 h.(3)The fusion protein with His tag was purified by Ni-NTA column with biological activity,the thermal hysteresis activity of protein(at 4.0 mg/mL)was-0.4? and the yield of target protein reached 56 mg/L.(4)Directed evolution technique was successfully applied to antifreeze protein and a large number of mutants was screened,a good mutant was found,with osmolality increased by 8.4%comparing with the original protein and the thermal hysteresis activity of the TmAFP protein(at 4.0 mg/mL)reached-0.6?.