School of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung, 40402Taiwan, Republic of China.
BMC Biotechnol. 2012 Jun 6;12:27. doi: 10.1186/1472-6750-12-27.
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.
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.
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.
禽传染性贫血病毒（CAV）VP3 基因编码的凋亡蛋白（Apoptin）不仅能诱导前体胸腺细胞凋亡，还能特异性杀伤人癌细胞、肿瘤细胞和转化细胞，而不影响正常细胞的增殖。该蛋白的这种肿瘤特异性凋亡特性可能使其成为一种具有肿瘤治疗应用前景的蛋白药物。然而，由于表达量低和蛋白溶解性差等几个主要问题，在细菌中生产凋亡蛋白一直受到阻碍。
利用大肠杆菌表达系统，显著提高了源自禽传染性贫血病毒的全长重组凋亡蛋白的表达量。CAV VP3 基因与含有转录激活因子（TAT）蛋白转导结构域（PTD）的合成序列融合。构建的融合蛋白被克隆到不同的表达载体中，并在不同的大肠杆菌菌株中表达。当 TAT-Apoptin 与 GST 标签融合而不是 His 标签融合时，TAT-Apoptin 在大肠杆菌中的表达量显著增加。对凋亡蛋白的各种稀有氨基酸密码子进行优化后，在大肠杆菌 BL21（DE3）中 GST-TAT-Apoptin（opt）的表达水平进一步显著提高。在 25°C 下用 0.1mM IPTG 诱导 4 小时后，细菌培养物的最高蛋白表达量达到 8.33g/L。此外，在这些条件下表达的约 90%的 GST-TAT-Apoptin（opt）是可溶性的。通过 GST 亲和层析纯化后，用纯化的重组 TAT-Apoptin（opt）蛋白评估重组蛋白对肿瘤细胞的凋亡活性。结果表明，在大肠杆菌中表达的 GST-TAT-apoptin（opt）具有凋亡活性，能够诱导人早幼粒细胞白血病 HL-60 细胞进入凋亡。
与 GST 标签融合并优化密码子的表达纯化重组 TAT-Apoptin（opt）具有很大的潜力。这种蛋白将来可能会开发出一种能够特异性杀伤肿瘤细胞的治疗性蛋白。
