Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5 alpha under the control of the Bacillus BsXA promoter

Xylanase A (XynA) is a class G/11 xylanase secreted by Bacillus subtilis. XynA was purified to homogeneity from B. subtilis strain 168 culture supernatants by ethanol precipitation and cation-exchange chromatography. The DNA fragment encoding the XynA together with the BsXA promoter region was amplified by PCR from B. subtilis 168 genomic DNA, and cloned into the plasmid pT7T3 to give the plasmid pT7BsXA. After transformation of Escherichia coli DHSa with pT7BsXA, a 19-fold increase in the levels of the secreted XynA was detected in the supernatant as compared with the B. subtilis culture. Correct post-translation modification of the recombinant protein was confirmed by N-terminal amino acid sequencing and MS analyses. The pH- and temperature-dependences of the native and recombinant proteins were identical, indicating that the pT7BsXA may be useful for the constitutive expression of heterologous protein in E. call.