The Human OX40 Fusion Proteins Expression and Purification

Objective To optimize the expression of the fusion protein with the human's OX40 extracellular segment,and purify the interest protein. So as to construct a solid basis for preparing the monoclonal antibody of human OX40 and further studying. Methods ① The extracellular pET32a-OX40 recombinant plasmid sequenced correctly was transfected into E. coli. BL21 ( DE3) ,a bacteria strain for indu- cing expression. ②After the positive bacteria was induced by IPTG in proper conditions, the product of the fusion protein and its location in the cell were analyzed by SDS-PAGE and then the best expression condition was determined. ③The interest protein was purified by Ni Sepha- rose and used UF lube to desalt the protein. Then counted out the concentration of fusion protein. Results ① The positive stain that ex- pressed the pET32a-OX40 were obtained successfully. ②SDS-PAGE showed a new protein in BL21/pET32a-OX40 after IPTG induction. Its relative atomic weight was between 31.0kD and 42. 7kD. The new protein existed not only in inclusion body of bacterial but also in superna- tant, mainly in inclusion body. ③The concentration oi fusion protein was 56. 266g/L. Conclusion A fusion protein of OX40 extracellular segment,which expressed pET32a-OX40,was successfully induced by SDS-PAGE. The pure protein have been obteined by Ni Sepharose.