Prokaryotic Expression And Purification Of The Soybean 7S Protein ?-Subunit Core Region

Soybean,as a food with relatively comprehensive nutritional components,has broad development prospects.As an important component of soybean protein,the physicochemical properties of 7S glycinin domain will affect the functional properties of soybean protein,which in turn will affect the quality of soybean products.Therefore,exploring 7S soybean globulin at the molecular level is helpful for breakthroughs in the production practice and theoretical research of soybean and its products.At present,researchers have found that the glycosyl group and the extension area have an important influence on the function of 7S glycinin,but the related research is not comprehensive.In this study,the preparation of recombinant 7S glycinin subunits with extended regions and N-linked glycosyl deletions helps to study the effects of different domains on the functional properties of 7S globulins,and lays the foundation for the construction of recombinant soybean protein water dispersion system.The results obtained in this paper are as follows:The total RNA of soybean was extracted from Qihuang 34 soybean variety of Shandong Academy of Agricultural Sciences,and its c DNA single strand was constructed by reverse transcription.Design primers F1/R1 according to the sequence of the?-subunit core region(GB number NM?001249927.2)found on the NCBI official website,and obtain the target gene?-subunit core region by RT-PCR amplification.The length of the obtained fragment is about 1300 bp,which is consistent with the theoretical value.Then it is connected to the p GEM-T Easy vector and introduced into the competent cell E.coli DH5?.After blue and white spot screening,colony PCR,Eco R?/Xho?double digestion,sequence determination and comparison screen to obtain the correct recombinant cloning vector,named p GEM-?_c.Recombinant cloning vector and plasmid p ET-28a were double-digested respectively and ligated,and then introduced into competent cells E.coli BL21(DE3).The double-digestion,colony PCR and sequence comparison were used to screen the correct recombinant expression vector,named p GEM-28a-?_c.When the positive engineering bacteria p GEM-28a-?_c-BL21 was cultured until the OD600 of the bacterial solution was0.8,0.2 mmol/L of the inducer IPTG was used to continue the culture at 37?for 9 hours to obtain recombinant?-subunit core region protein with high expression and molecular weight of about 50 k Da.Ultrasound was used to disrupt the bacteria for 4-5 cycles under the conditions of amplitude of 25,6 s/4 s and 30 min.Centrifugation was performed at 8500 r/min for 45minutes at 4?and the supernatant was collected.Apply the collected supernatant to a protein purification system with a nickel ion column.The flow rates of the equilibrium solution and the eluent were both 1 m L/min,and the eluent at a concentration of 20%was used to elute the impurities in the column.The 80%eluent elutes the recombinant?-subunit core region protein,and its content can reach more than 87%.