Beyreuther K, Adler K, Fanning E, Murray C, Klemm A, Geisler N
Eur J Biochem. 1975 Nov 15;59(2):491-509. doi: 10.1111/j.1432-1033.1975.tb02477.x.
The lac repressor from Escherichia coli, composed of four identical subunits with a molecular weight of 37160, was carboxymethylated and fragmented by tryptic digestion and cyanogen bromide treatment. Using ion-exchange chromatography, gel filtration and preparative thin-layer electrophoresis and chromatography 29 of the 30 tryptic peptides were isolated in pure form. Direct Edman degradation and the dansyl-Edman technique were used to determine the sequence of the small tryptic peptides. Special emphasis was put on the sequence determination of the six large tryptic fragments which together account for 177 residues, corresponding to 51% of the repressor subunit with its 347 residues. The large tryptic fragments were analyzed after fragmentation with chymotrypsin, thermolysin and dipeptidyl aminopeptidase I. Thus the sequence of all 30 tryptic peptides could be deduced. The complete sequences of all cyanogen bromide fragments were deduced from peptides obtained by tryptic, chymotryptic and thermolytic digestion of the individual fragments and by automated stepwise Edman degradation of lac repressor and of the large cyanogen bromide fragments. The order of the cyanogen bromide fragments was given by overlapping tryptic peptides. The resulting amino acid composition of the monomer is Asp15, Asn11, Thr18, Ser30, Glu14, Gln27, Pro13, Gly22, Ala44, Cys3, Val33, Met9, Ile17, Leu40, Tyr8, Phe4, Trp2, Lys11, His7, Arg19. The sequence of lac repressor shows no similarities with that of other proteins known to bind to DNA or RNA. The N-terminal 55 residues contain two homologous regions. This part of the sequence which is involved in lac operator binding might have been formed by gene duplication.
来自大肠杆菌的乳糖阻遏蛋白由四个分子量为37160的相同亚基组成，经羧甲基化处理后，通过胰蛋白酶消化和溴化氰处理进行片段化。利用离子交换色谱、凝胶过滤以及制备型薄层电泳和色谱法，30个胰蛋白酶肽段中的29个以纯形式被分离出来。采用直接埃德曼降解法和丹磺酰-埃德曼技术来确定小胰蛋白酶肽段的序列。特别着重于对六个大的胰蛋白酶片段进行序列测定，这六个片段总共包含177个残基，相当于具有347个残基的阻遏蛋白亚基的51%。在用胰凝乳蛋白酶、嗜热菌蛋白酶和二肽基氨肽酶I进行片段化后，对这些大的胰蛋白酶片段进行了分析。由此可以推导出所有30个胰蛋白酶肽段的序列。所有溴化氰片段的完整序列是通过对各个片段进行胰蛋白酶、胰凝乳蛋白酶和嗜热菌蛋白酶消化所得到的肽段，以及通过对乳糖阻遏蛋白和大的溴化氰片段进行自动逐步埃德曼降解推导出来的。溴化氰片段的顺序由重叠的胰蛋白酶肽段确定。所得单体的氨基酸组成为：天冬氨酸15个、天冬酰胺11个、苏氨酸18个、丝氨酸30个、谷氨酸14个、谷氨酰胺27个、脯氨酸13个、甘氨酸22个、丙氨酸44个、半胱氨酸3个、缬氨酸33个、甲硫氨酸9个、异亮氨酸17个、亮氨酸40个、酪氨酸8个、苯丙氨酸4个、色氨酸2个、赖氨酸11个、组氨酸7个、精氨酸19个。乳糖阻遏蛋白的序列与已知的其他与DNA或RNA结合的蛋白质序列没有相似性。N端的55个残基包含两个同源区域。序列中参与乳糖操纵基因结合的这一部分可能是由基因复制形成的。
