Majumdar A, Rudikoff S, Adhya S
J Biol Chem. 1987 Feb 15;262(5):2326-31.
The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31. The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing. Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor. The protein has been purified to homogeneity in three steps. The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM. The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val-. Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off. The repressor is present in solution as a dimer of a 37-kDa subunit. The protein binds to gal DNA containing wild type and not mutant operator sequences. As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon. Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment. Experiments to study the mechanism of repressor action are discussed.
编码大肠杆菌中半乳糖阻遏蛋白的galR基因，已与质粒pKC31中噬菌体λ的强pL启动子融合。pL启动子被温度敏感型λ阻遏物CIts857抑制，以防止细胞死亡。对pL-galR融合质粒进行热诱导可合成大量活性半乳糖阻遏蛋白。该蛋白已通过三个步骤纯化至同质。在盐浓度低于200 mM时，活性阻遏物在粗提取物中可逆不溶，这极大地有助于纯化。通过自动Edman降解确定的氨基末端氨基酸序列为：N-丙氨酸-苏氨酸-异亮氨酸-赖氨酸-天冬氨酸-缬氨酸-丙氨酸-精氨酸-亮氨酸-丙氨酸-甘氨酸-缬氨酸-丝氨酸-缬氨酸-丙氨酸-苏氨酸-缬氨酸-。将该序列与从galR基因的DNA序列推导的序列进行比较，结果表明，第1位丙氨酸之前的甲酰甲硫氨酸残基被切除。阻遏物以37 kDa亚基的二聚体形式存在于溶液中。该蛋白与含有野生型而非突变操纵序列的半乳糖DNA结合。正如所预测的，D-半乳糖或D-岩藻糖的存在会抑制这种序列特异性结合，这两种糖都是半乳糖操纵子的体内诱导剂。半乳糖阻遏蛋白通过与位于半乳糖启动子区段两侧但不重叠的两个空间分离的操纵子结合来抑制半乳糖操纵子的表达。文中讨论了研究阻遏物作用机制的实验。
