Heterogeneous expression of DnaK gene from Alicyclobacillus acidoterrestris improves the resistance of Escherichia coli against heat and acid stress
Alicyclobacillus acidoterrestris, an acidophilic and thermophilic bacteria, is an important microbial resource for stress resistance genes screening. In this study, DnaK gene from A.
acidoterrestris was subcloned to construct the recombinant plasmid pET28a–DnaK. The successful construction of the plasmid was verified by double-enzyme digestion and sequencing analysis.
The recombinant plasmid was transformed into Escherichia coli BL21 and isopropy-?-d-thiogalactoside (IPTG) was used to induce recombinant E. coli to express DnaK gene.
A 70 kD fusion protein was identified by SDS-PAGE, which suggested that DnaK gene from A. acidoterrestris was successfully expressed.
The recombinant and wild BL21 were treated with high temperatures of 54, 56 and 58 °C at pH values of 5.0–7.0 to compare the effects of heterogeneous expression of the DnaK gene from A. acidoterrestris on the stress resistance.
The experimental results showed that survival rate of recombinant BL21–DnaK has been improved considerably under heat and acid stresses in contrast with the wild BL21, and D-values of recombinant BL21 were 14.7–72% higher than that of wild BL21, which demonstrated that heterogeneous expression of DnaK gene from A. acidoterrestris could significantly enhance the resistance of host bacteria E.