Isolation And Purification Of Soybean 7S Globulin Subunit

7S globulin and 11 S globulin are the major components of soybean protein.The gel formation ability of 11 S globulin is stronger than 7S globulin.Research showed that glycosyl structure in 7S globulin can affect its gel formation.The subunit gene of 7S globulin is transferred into the E.coli expression system,and the recombinant subunit can be obtained.The recombinant subunit lacks the glycosyl structure compared with the natural 7S globulin subunit.In this study,the 7S globulin subunit and the recombinant subunit were isolated and purified and the relationship between the 7S subunit structure and its functional properties was revealed.Using Lu 96150 soybean seeds as raw materials,7S globulin can be isolated by isoelectric precipitation.The extracted 7S globulin was further purified by agarose gel CL-6B.The purification conditions were optimized by adjusting the loading amount,loading speed and loading concentration.Results: When the sample was more than 200 mg,the loading concentration was 100mg/ml and the loading speed was 0.1ml/min,the purification of the sample is the best.In addition,ammonium sulfate precipitation is also commonly used method for protein separationthe.The purity of 7S glycinin extracted after the isoelectric point precipitation method and ammonium sulfate precipitation method(81.8%)was comparable to that of 7S globulin using gel chromatography(84.2%).As 7S glycinin is a trimer consisting of hydrophobic interactions of ??,? and ? subunits.and the three subunits were homologous to some extent,so the 7S globulin was denatured with a strong denaturant urea treatment,to make free the three subunits,then the three subunits were separated using DEAE Fast Flow agarose gel FF column.The separation conditions of the subunits were screened by adjusting the loading amount when the loading speed was 0.1ml/min and the loading concentration was 100mg/ml.Results: When the loading concentration was 100mg/ml,the loading rate was 0.1ml/min,and the amount of sample was about 450 ~ 500 mg could get the best separation results and lest miscellaneous protein peaks.A recombinant protein ?? subunit about 70 kD was well expressed by E.coli pET-28a-??-BL21 inducing with IPTG.After ultrasonic crushing,centrifugation,we can getthe rough recombinant ?? subunit.The recombinant protein was further purified by the Ni2 +affinity chromatography column using AKTA protein purification system.The purification conditions were optimized by adjusting the flow rate of the equilibrium solution using gradient elution.Results: When the Ni2+ column was equilibrated with an equilibrium solution at a flow rate of 5 ml/min,followed by a linear gradient elution method,a higher purity of89.1% of target protein could be obtained.