IDENTIFICATION OF A NOVEL SUGAR-H+ SYMPORT PROTEIN, FUCP, FOR TRANSPORT OF L-FUCOSE INTO ESCHERICHIA-COLI
IDENTIFICATION OF A NOVEL SUGAR-H+ SYMPORT PROTEIN, FUCP, FOR TRANSPORT OF L-FUCOSE INTO ESCHERICHIA-COLI
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DOI：
10.1111/j.1365-2958.1994.tb01066.x
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发表时间：
1994-06-01
影响因子：
3.6
通讯作者：
HENDERSON, PJF
中科院分区：
文献类型：
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作者：
GUNN, FJ;TATE, CG;HENDERSON, PJF
L-岩藻糖(6-脱氧-L-半乳糖)是许多微生物唯一的碳源，其转运是由L-岩藻糖-H+结合活性介导的。为了确定大肠杆菌FucP基因编码的转运蛋白的性质并鉴定其蛋白产物，将其克隆在可诱导的T7RNA聚合酶和lambda OLPL启动子的下游。T7启动子的诱导导致[C-14]-L-岩藻糖摄取活性的表达和[S-35]-蛋氨酸标记的32 kDa蛋白的表达，其水平太低，无法通过考马斯亮蓝染色或蛋白质测序检测到。通过对lambda OLPL启动子的诱导，出现了L-岩藻糖-H+共转运蛋白活性和高水平表达的考马斯亮蓝染色的32 kDa膜蛋白，经N-末端蛋白测序证实为FucP。因此，FucP蛋白是一种在氨基酸序列上不同于任何其他已知转运蛋白的糖-H+转运蛋白。这些和其他结果说明了尝试放大表达膜运输蛋白的克隆策略的一般不可预测性。
L-Fucose (6-deoxy-L-galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia cell is mediated by an L-fucose-H+ symport activity. In order to determine the nature of a putative transporter encoded by the E. coli fucP gene and identify its protein product it was cloned downstream of the inducible T7 RNA polymerase and lambda OLPL promoters. Induction of the T7 promoter resulted in the expression of [C-14]-L-fucose uptake activity and the concomitant expression of a [S-35]-Met-labelled 32 kDa protein at levels too low for detection by staining with Coomassie brilliant blue or for protein sequencing. Induction of the lambda OLPL promoter caused the appearance of L-fucose-H+ symport activity and of a Coomassie brilliant blue-stained 32 kDa membrane protein expressed at high levels sufficient for Identification as FucP by N-terminal protein sequencing. The FucP protein is, therefore, a sugar-H+ symporter different in amino acid sequence from any other known transporter. These and other results illustrate the general unpredictability of cloning strategies for attempting the amplified expression of membrane transport proteins.