Efficient constitutive expression of Bacillus subtilis xylanase A in Escherichia coli DH5alpha 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 DH5alpha 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. coli.
Departamento de Biologia Molecular e Celular e Bioagentes Patogênicos, FMRP-USP (Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo), Ribeirão Preto, SP, Brazil.
Roberto
R
Ruller
José César
JC
Rosa
Victor M
VM
Faça
Lewis J
LJ
Greene
Richard J
RJ
Ward
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000032:analysis; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013696:Temperature
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases