Efficient production of an engineered apoptin from chicken anemia virus in a recombinant E. coli for tumor therapeutic applications.

BACKGROUND

Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria.

RESULTS

Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptin(opt) in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptin(opt) under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptin(opt) protein was used to evaluate the recombinant protein's apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptin(opt) showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.

CONCLUSIONS

On expression in E. coli, purified recombinant TAT-Apoptin(opt) that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.