Ruller Roberto, Rosa José César, Faça Victor M, Greene Lewis J, Ward Richard J
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.
Biotechnol Appl Biochem. 2006 Jan;43(Pt 1):9-15. doi: 10.1042/BA20050016.
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.
木聚糖酶A（XynA）是一种由枯草芽孢杆菌分泌的G/11类木聚糖酶。通过乙醇沉淀和阳离子交换色谱法从枯草芽孢杆菌168菌株的培养上清液中纯化得到了均一的XynA。编码XynA的DNA片段与BsXA启动子区域一起通过PCR从枯草芽孢杆菌168基因组DNA中扩增出来，并克隆到质粒pT7T3中，得到质粒pT7BsXA。用pT7BsXA转化大肠杆菌DH5α后，与枯草芽孢杆菌培养物相比，在上清液中检测到分泌的XynA水平增加了19倍。通过N端氨基酸测序和质谱分析证实了重组蛋白的正确翻译后修饰。天然蛋白和重组蛋白的pH和温度依赖性相同，表明pT7BsXA可用于在大肠杆菌中组成型表达异源蛋白。
