CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF THE KLEBSIELLA-PNEUMONIAE GENES FOR PRODUCTION, SURFACE LOCALIZATION AND SECRETION OF THE LIPOPROTEIN PULLULANASE
CLONING AND EXPRESSION IN ESCHERICHIA-COLI OF THE KLEBSIELLA-PNEUMONIAE GENES FOR PRODUCTION, SURFACE LOCALIZATION AND SECRETION OF THE LIPOPROTEIN PULLULANASE
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DOI：
10.1002/j.1460-2075.1987.tb02679.x
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发表时间：
1987-11-01
期刊：
影响因子：
11.4
通讯作者：
PUGSLEY, AP
中科院分区：
文献类型：
--
作者：
DENFERT, C;RYTER, A;PUGSLEY, AP
本文描述了在大肠杆菌中重建异源蛋白质分泌系统，该系统包含胞外蛋白质基因及其同源分泌基因。所涉及的蛋白质，普鲁兰酶，是革兰氏阴性细菌肺炎克雷伯氏菌的分泌脂蛋白。它最初定位于细胞表面，然后特异性地释放到培养基中。E. pullA基因在大肠杆菌中表达，但不表达或分泌。将pullA基因与pullA基因一起克隆到一个18.8kbp的片段中。pneumoniae染色体DNA E.该片段在大肠杆菌中的表达、暴露和分泌都是由麦芽糖诱导的。转座子突变表明分泌基因位于pulA的两侧。位于pulA 5“的分泌基因在先前鉴定的malT调节的malX启动子的控制下以与pulA相反的方向转录。因此，这些分泌基因是麦芽糖调节子的一部分，因此与pulA共表达。转座子诱变表明，位于pulA 3“的分泌基因不与pulA共转录，提高了一些分泌功能不受麦芽糖调节的可能性。
This article describes the reconstitution in Escherichia coli of a heterologous protein secretion system comprising a gene for an extracellular protein together with its cognate secretion genes. The protein concerned, pullulanase, is a secreted lipoprotein of the Gram-negative bacterium Klebsiella pneumoniae. It is initially localized to the cell surface before being specifically released in to the medium. E. coli carrying the cloned pullulanase structural gene (pulA) produces pullulanase but does not expose or secrete it. Secretion genes were cloned together with pulA in an 18.8 kbp fragment of K. pneumoniae chromosomal DNA. E. coli carrying this fragment exhibited maltose-inducible production,exposition and specific secretion of pullulanase. Transposon mutagenesis showed that the secretion genes are located on both sides of pulA. Secretion genes located 5'' to pulA were transcribed in the opposite orientation to pulA under the control of the previously identified, malT-regulated malX promoter. Thus these secretion genes are part of the maltose regulon and are therefore co-expressed with pulA. Transposon mutagenesis suggested that secretion genes located 3'' of pulA are not co-transcribed with pulA, raising the possibility that some secretion functions are not maltose regulated.