%0 Journal Article
%T Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications
%A Meng-Shiou Lee
%A Fang-Chun Sun
%A Chi-Hung Huang
%A Yi-Yang Lien
%A Shin-Huei Feng
%A Guan-Hua Lai
%A Meng-Shiunn Lee
%A Jung Chao
%A His-Jien Chen
%A Jason T C Tzen
%A Hao-Yuan Cheng
%J BMC Biotechnology
%D 2012
%I BioMed Central
%R 10.1186/1472-6750-12-27
%X 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-Apoptinopt 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£¿¡ãC. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein¡¯s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.On expression in E. coli, purified recombinant TAT-Apoptinopt 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.
%U http://www.biomedcentral.com/1472-6750/12/27/abstract