Optimization of expression JTAT protein with emphasis on transformation efficiency and IPTG concentration

Publication date: December 2017Source: Journal of Genetic Engineering and Biotechnology, Volume 15, Issue 2Author(s): Endang Tri Margawati, Asrul Muhamad Fuad, Indriawati, Muhamad Ridwan, Slamet Diah VolkandariAbstractOne 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 ...
Source: Journal of Genetic Engineering and Biotechnology - Category: Biotechnology Source Type: research