Margawati Endang Tri, Fuad Asrul Muhamad, Ridwan Muhamad, Volkandari Slamet Diah
Research Center for Biotechnology, The Indonesian Institute of Sciences (LIPI), Jalan Raya Bogor KM. 46, Cibinong 16911, Indonesia.
J Genet Eng Biotechnol. 2017 Dec;15(2):515-519. doi: 10.1016/j.jgeb.2017.06.009. Epub 2017 Jul 6.
One of small accessory genes between and is gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing () in advance using adopted methods of M1 (MgCl + CaCl) and M2 (CaCl + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic gene encoding protein JTAT was previously cloned into pBT-. Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD = 4), incubated for 3 h. Pellets were harvested by centrifugation 4000 rpm; 4 °C; 15 min. Buffer B (10 mM Immidazole) was added into pellets, lysed by freeze-thaw followed by sonication. Supernatant was collected by centrifugation (10,000 rpm; 4 °C; 20 min) and purified using Ni-NTA Agarose resin, released by elution buffer (E) containing 400 mM Immidazole to collect purified protein twice (E1, E2). The protein was characterized by SDS-PAGE and Western Blot (WB), quantified (at 595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 10) than M1 (3.10 × 10). The JTAT protein was expressed at a right size of 11.8 kDa. Concentration of 200 µM IPTG produced a significantly better protein yield (1.500 ± 0.089 mg/ml; < 0.05) than 600 µM IPTG (0.896 ± 0.052 mg/ml) and not different to 400 µM IPTG (1.298 ± 0.080 mg/ml). This research indicated that transformation efficiency needs to be taken account in prior of optimization of the protein expression.
和之间的一个小辅助基因是编码TAT蛋白的基因。本研究旨在通过采用M1（MgCl₂ + CaCl₂）和M2（CaCl₂ + 甘油）方法预先制备（）来优化Jembrana TAT（JTAT）蛋白的表达。由更好的转化方法产生的最佳转化效率用于后续JTAT蛋白的表达。先前已将编码JTAT蛋白的合成基因克隆到pBT-中。将200、400、600 μM IPTG诱导用于小体积培养（200 ml；OD = 4），孵育3小时。通过4000 rpm、4°C、15分钟离心收集沉淀。向沉淀中加入缓冲液B（10 mM咪唑），通过冻融然后超声处理进行裂解。通过离心（10,000 rpm、4°C、20分钟）收集上清液，并使用Ni-NTA琼脂糖树脂进行纯化，用含有400 mM咪唑的洗脱缓冲液（E）洗脱以收集两次纯化的蛋白（E1、E2）。通过SDS-PAGE和Western Blot（WB）对蛋白进行表征，事先用BSA标准方法（在595 nm处）进行定量。结果表明，M2（2.53×10）的转化效率优于M1（3.10×10）。JTAT蛋白以11.8 kDa的正确大小表达。200 μM IPTG的浓度产生的蛋白产量（1.500±0.089 mg/ml；P < 0.05）明显优于600 μM IPTG（0.896±0.052 mg/ml），与400 μM IPTG（1.298±0.080 mg/ml）无差异。本研究表明，在优化蛋白表达之前需要考虑转化效率。
