Fig. 3
DHFR production using cell-free expression system. a DHFR expression in the presence or absence of PrS2. Solutions A and B were mixed according to the manufacturer’s protocol (NEB), and the mixture was incubated as a negative control (lane 1), and DNA(10 ng/μl) of DHFR were added and incubated as a positive control (lane 2), DNA of DHFR (10 ng/μl) together with 0.2 mg/ml of PrS2 (10 μM) were incubated. All incubations were performed at 37 °C for 2 h. After the incubation, 2× SDS loading dye was added and the mixture was subjected to the 17 % of SDS-PAGE and the molecular weight marker was shown as lane M; b DHFR expression in the presence of PrS2-Bac7 with and without factor Xa. Solutions A and B from the Cell-Free system (NEB) were mixed, the reaction mixture without DNA was used as a negative control (lane 1) and the reaction mixture in the presence of DNA (10 ng/μl) but in the absence of proteins was used as a positive control (lane 2). Lane 3, the reaction mixture containing DNA (10 ng/μl) together with 10 μM of Bac7 (1–16) as a control; lane 4, the reaction mixture containing DNA (10 ng/μl) together with 10 μM of PrS2-Bac7 (1–35) after cleavage by Factor Xa; lane 5, the reaction mixture containing DNA (10 ng/μl) together with 10 μM of PrS2-Bac7 (1–35) without cleaving by Factor Xa, and lane M, molecular weight marker. All the reactions were carried out at 37 °C for 2 h, After the reaction, 2× SDS loading buffer was added to the reaction mixture, which was then subjected to 15 % SDS-PAGE, followed by Coommassie blue staining