Improving soluble expression of a single-chain antibody Pertuzumab by co-expression of chaperones

Introduction: On September 30, 2013, the FDA granted accelerated approval to pertuzumab for use in combination with trastuzumab and docetaxel as neoadjuvant treatment of patients with HER2-positive, Also, One of the trademarks of trastuzumab was introduced as the third Best-selling Cancer Drug in 2018.In previous studies, the majority of pertuzumab antibody was expressed as inclusion bodies by Escherichia coli. Various methods have been proposed to stop or reduce the formation of inclusion bodies during the production of recombinant proteins. One of them is the co-expression of molecular chaperones with these proteins. Methods: The mixture of plasmids pKJE7 and pET22b-ScFv was transformed by the electroporation method to the cells of the E. coli strain BL21(DE3). Bacteria harboring chaperone and ScFv gene were cultured and when the culture reached to OD600 of 0.5, arabinose was added to induce expression chaperone and IPTG (1mM) was used to express the ScFv protein. Then, bacterial cells were harvested and dispersed in a suitable buffer, and the cells were lysed by sonication. The soluble and insoluble fractions of cell lysate were separated by centrifugation and each part was purified using a Ni-NTA affinity column. To compare the expression level antibodies were used in each system of expression by SDS-PAGE and Western blotting assay, as well as the method of Bradford to determine protein concentration. Results: Our results showed that the majority (80-90 %) of anti-her2his-ScFv was expressed as inclusion bodies and insoluble protein. however, co-expression of chaperones and anti-her2his-ScFv significantly increase soluble expression of anti-her2his-ScFv. Conclusion: The results obtained in the present study indicate that co-expression of molecular chaperones pKJE7 will enhance solubility and as result the biological function of anti-her2his-ScFv expressed by E. coli.

Keywords:

HER2 , Single-chain variable fragment , Molecular chaperone , Inclusion bodies , Protein folding