Prokaryotic Expression And Purification Of The Soybean 7S Protein α’-Subunit

In order to obtain the N-linked glycans lacked a’subunit of soybean 7S protein. The total RNA were extracted from the seeds of soybean variety Lu 96150. The a’subunit and its core region a’c coding sequence were amplified by RT-PCR one step assay, and then inserted into the vector of pGEM-T Easy to construct the recombinant cloning vectors pGEM-a’ and pGEM-a’c. After digested by XhoI/EcoRI the a’fragment was inserted into the prokaryotic expression vector pET28a containing His-tag. The recombinant expression vector pET-28a-a’ was verified by the colony PCR, restriction endonuclease digestion and DNA sequencing. Then the pET-28a-ar vector was transformed into E.coli host strain BL21 (DE3) for IPTG induction expression. The induced expression conditions of the recombinant a’subunit were optimized. The recombinant a’subunits was purified by the nickel ion affinity chromatography column and the conditions of purification were screened, the results are as follows.The size of the target genes a’and a’c was consistent with the theory, which is 1700 bp and 1300 bp respectively. The recombinant cloning vector and the recombinant experssion vector were successfully consturcted named pGEM-a’, pGEM-a’c and pET-28a-a’ respectively. A recombinant protein a’subunit about 70ku was well expressed reaching up to 31.6% by pET-28a-a’-BL21 inducing with 0.2mmol/L IPTG at 30℃ for 9h and the OD value was 0.8. After ultrasonication and centrifugation a part of the target protein a’was found in the supernatant not in the form of inclusion body, more convenient to the further purification work.The recombinant a’subunit was purified by the nickel ion affinity chromatography colum. We found that 5.0mL/min binding buffer could reduce the target protein content in the Fi protein peak and improve the efficiency of purification. The 30% and 35% concentration of the eluent buffer could remove a lot of contaminant protein in the chromatography column. Washing with the 50% concentration of the eluent buffer the recombinant a’subunit protein content reached 53% . Then washing with 80% and 100% concentration of the elution buffer the recombinant a’subunit protein content both could reach more than 80%.