Fig. 4

Relative expression of eng mRNA in P. pastoris CL2 in response to cold-shock at 4 °C. CL2 cells were grown in medium BMGH (30 °C for 4 h), from OD₆₀₀ = 1 inoculum, then subjected to cold-shock for the induction of eng mRNA transcript expression (4 °C for 6 h). Samples were taken at 0, 2 and 6 h for analysis by qRT-PCR; and finally, they were transferred to 30 °C for 4 h for ENG product synthesis, stage in which the corresponding sample at 2 h was taken for analysis by qRT-PCR. The values of relative expression of eng mRNA were calculated in relation to eng and the housekeeping constitutive gene act1 (control) expression using method 2^−ΔΔCt