Prokaryotic Expression And Purification Of ?-subunit Of ?-conglycinin

Soybean protein is an important source of nutrients for living organisms,and the development of its potential nutritional health benefits requires research at the subunit level.The separation of different subunits of soybean protein isolates is limited by the high cost of purification column,long labor time,small preparation amount and low purity.Exploring the function of different subunits at the molecular level is at the forefront of research.In order to solve the problem that different subunits of ?-conglycinin were difficult to prepare and could not be obtained in large quantities,this study selected"Qihuang 34"soybeans,and used gene cloning technology and prokaryotic expression system to encode the ?-subunits of ?-conglycinin.The affinity chromatography method was adopted to obtain the target protein with higher purity through the protein purification system.The results are as follows:The total RNA of soybean was extracted,and the ?-subunit gene sequence of ?-conglycinin was amplified by PCR method,which was cloned into pGEM-T Easy vector and named pGEM-T Easy-?.Then,the ?-subunit gene sequence of?-conglycinin on pGEM-T Easy-?vector was digestised and connected to p ET-28a expression vector,which was named pET-28a-?.Introducing the pET-28 a-?into E.coli BL21(DE3)to obtain the positive clone strain pET-28a- ?-BL21.The expression of recombinant ?-subunit protein was optimized by different combinations of conditions.The recombinant ?-subunit protein increased at a high level when the concentration of the bacteria solution(OD₆₀₀value)reached 0.6 and the protein was cultured at 30? for 9h,which was 25.83%of the total protein expressed in E.coli.Under these conditions,protein purity was increased by 10%?15%compared with 37?,OD₆₀₀concentration(0.8)and induction culture for 9 hours adopted in most studies.The molecular weight of the ?-subunit protein was about 70 k Da.Linear elution was carried out to determine the peak position of ?-subunit protein at about 70% imidazole volume fraction at first.Since the protein electrophoresis results showed doping with other proteins,distribution elution was adopted to improve the protein purity.The final purification conditions of the experiment were as follows:purification were carried out using a nickel affinity chromatography column with elution of phosphate buffer containing 350 mmol/L imidazole.The purity of recombinant protein ?-subunit in elution peak was about 70.0%,and the weight can be obtained in milligram-scale.This experiment lays the foundation for the later study of the structure and physicochemical properties of the ?-subunit of ?-conglycinin.