Real-time monitoring of rhamnose induction effect on the expression of mpt64 gene fused with pelB signal peptide in Escherichia coli BL21 (DE3).

In this research, Escherichia coli BL21 (DE3) harboring an expression vector constructed with a rhamnose-inducible promoter and a pelB signal peptide was used as a host cell to produce MPT64 protein. The objective of this research was to figure out the optimum time of mpt64 gene expression through real-time monitoring of MPT64 protein production and distribution in host compartments. The mpt64 expression was regulated by the rhamnose presence at a concentration of 4 mM. The real-time isolated protein was monitored using polyacrylamide gel electrophoresis in denaturation condition. Based on real-time monitoring, the MPT64 protein (24 kDa) in the cytoplasm was optimum detected at 24 h after induction. For periplasmic fraction, the protein was detected at 4 h after induction but thinning at 15 h after induction. At 16 h after induction, the MPT64 protein band was found in the medium with increasing concentrations until 24 h. Thus, it can be concluded that the mpt64 gene expression was regulated in the presence of rhamnose as an inducer, and the proteins were shown to be translocated throughout the host cell compartment with different levels of protein accumulation at different times, according to the role of pelB as a signal peptide.

Optimization of culture conditions for Mpt64 synthetic gene expression in Escherichia coli BL21 (DE3) using surface response methodology.

MPT64 is a specific protein that is secreted by Mycobacterium tuberculosis complex (MTBC). The objective of this study was to obtain optimum culture conditions for MPT64 synthetic gene expression in Escherichia coli BL21 (DE3) by response surface methodology (RSM). The RSM was undertaken to optimize the culture conditions under different cultivation conditions (medium concentration, induction time and inducer concentration), designed by the factorial Box-Bhenken using Minitab 17 statistical software. From the randomized combination, 15 treatments and three center point repetitions were obtained. Furthermore, expression methods were carried out in the flask scale fermentation in accordance with the predetermined design. Then, the MPT64 protein in the cytoplasm of E. coli cell was isolated and characterized using sodium dodecyl sulfate polyacrilamide electrophoresis (SDS-PAGE) then quantified using the ImageJ program. The optimum conditions were two-fold medium concentration (tryptone 20 mg/mL, yeast extract 10 mg/mL, and sodium chloride 20 mg/mL), 5 h of induction time and 4 mM rhamnose. The average concentration of recombinant MPT64 at optimum conditions was 0.0392 mg/mL, higher than the predicted concentration of 0.0311 mg/mL. In conclusion, the relationship between the selected optimization parameters strongly influenced the level of MPT64 gene expression in E. coli BL21 (DE3).