Expression and characterization of bifunctional fusion proteins possessing antitumor and thrombolytic function for targeting therapy.

It is a usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. ΔSEC2, N-terminal deletion of 17 amino acids and C-terminal deletion of 132 amino acids, retained antitumor activity of SEC2. ΔSak, N-terminal deletion of 10 amino acids, had thrombolytic activity and specificity advantages. By utilizing bioactivities of ΔSEC2 and ΔSak, ΔSEC2-ΔSak and ΔSak-ΔSEC2 were constructed. Octreotide is a tumor targeting peptide and it can be combined with somatostatin (SST) receptors of tumor surface in ligand-receptor binding way. It can be used to increase specificity for tumor therapy. Based on previous studies, DNA sequence encoding octreotide gene was inserted into plasmid pET-28a-Δsec2-Δsak and pET-28a-Δsak-Δsec2. After expression and purification, fusion proteins could significantly stimulate proliferation of mouse spleen lymphocyte, obviously inhibit the growth of human gastric carcinoma BGC-823, and have thrombolytic activity, indicating that fusion proteins retained bioactivities of staphylococcal enterotoxin C2 and Sak. Furthermore, tumor binding capacity of fusion protein was confirmed through the coimmunoprecipitation method. The result showed that they could bind SST receptor 2 antibody, indicating that fusion proteins could be specifically targeted to tumor surface. It has important significance and may be used for targeted therapy.

Construction of novel chimeric proteins through the truncation of SEC2 and Sak from Staphylococcus aureus.

It is an usual clinical phenomenon that cancer patients are prone to thrombosis. Until now, there have been no efficient methods or appropriate drugs to prevent and cure tumor thrombus. Therefore, the construction of a bifunctional chimeric protein for the treatment of cancer, complicated with thrombosis, is of great significance. Utilizing the superantigenic activity of staphylococcal enterotoxin C2 (SEC2) and the thrombolytic activity of staphylokinase (Sak), Sak-linker-SEC2 and SEC2-linker-Sak were constructed which had good anti-tumor and thrombolytic activities at the same time. Due to the intrinsic emetic activity of SEC2 and high molecular weight (MW) of chimeric proteins (44 kDa), their clinical applications will be restricted. In this study, novel chimeric proteins including ΔSEC2-ΔSak and ΔSak-ΔSEC2 were constructed through the truncation of SEC2 and Sak without 9-Ala linker and His-tag. Compared with the former, both the truncated proteins preserved nearly the same anti-tumor and thrombolytic activities. In addition, their MWs were only 29 kDa and their immunoreactivities were slightly lower than that of Sak-linker-SEC2 and SEC2-linker-Sak, respectively. Therefore, the novel chimeric proteins possessed merits and characteristics, such as low MS, low immunogenicity, and difunctionality which the former had not. It will be of great interest if the above-mentioned proteins can be used to cure Trousseau syndrome in clinic.