| Autor: |
Endang Tri Margawati, Asrul Muhamad Fuad, Indriawati, Muhamad Ridwan, Slamet Diah Volkandari |
| Jazyk: |
angličtina |
| Rok vydání: |
2017 |
| Předmět: |
|
| Zdroj: |
Journal of Genetic Engineering and Biotechnology, Vol 15, Iss 2, Pp 515-519 (2017) |
| Druh dokumentu: |
article |
| ISSN: |
1687-157X |
| DOI: |
10.1016/j.jgeb.2017.06.009 |
| Popis: |
One of small accessory genes between pol and env is tat gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing Escherichia coli (E. coli) in advance using adopted methods of M1 (MgCl2 + CaCl2) and M2 (CaCl2 + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic tat gene encoding protein JTAT was previously cloned into pBT-hisC. Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD600 = 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 λ595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 106) than M1 (3.10 × 105). 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; P |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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