Gunn F J, Tate C G, Henderson P J
Department of Biochemistry, University of Cambridge, UK.
Mol Microbiol. 1994 Jun;12(5):799-809. doi: 10.1111/j.1365-2958.1994.tb01066.x.
L-Fucose (6-deoxy-L-galactose) is used as sole carbon source by many microorganisms, and its transport into Escherichia coli 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 [14C]-L-fucose uptake activity and the concomitant expression of a [35S]-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.
L-岩藻糖（6-脱氧-L-半乳糖）被许多微生物用作唯一碳源，其进入大肠杆菌的转运由L-岩藻糖-H⁺同向转运活性介导。为了确定大肠杆菌fucP基因编码的假定转运蛋白的性质并鉴定其蛋白质产物，将其克隆到可诱导的T7 RNA聚合酶和λ OLPL启动子的下游。T7启动子的诱导导致[¹⁴C]-L-岩藻糖摄取活性的表达以及[³⁵S]-甲硫氨酸标记的32 kDa蛋白质的伴随表达，其水平过低，无法通过考马斯亮蓝染色检测或进行蛋白质测序。λ OLPL启动子的诱导导致L-岩藻糖-H⁺同向转运活性的出现以及考马斯亮蓝染色的32 kDa膜蛋白的出现，该蛋白高水平表达，足以通过N端蛋白质测序鉴定为FucP。因此，FucP蛋白是一种糖-H⁺同向转运蛋白，其氨基酸序列与任何其他已知转运蛋白不同。这些结果以及其他结果说明了尝试扩增表达膜转运蛋白的克隆策略的普遍不可预测性。
