Brake A J, Fowler A V, Zabin I, Kania J, Müller-Hill B
Proc Natl Acad Sci U S A. 1978 Oct;75(10):4824-7. doi: 10.1073/pnas.75.10.4824.
A protein possessing both lac repressor and beta-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of beta-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of beta-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of beta-galactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and beta-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.
从大肠杆菌的一个缺失突变体中纯化出一种蛋白质，该蛋白质在一条约155,000道尔顿的单一多肽中同时具有乳糖阻遏物和β-半乳糖苷酶活性，在这个突变体中lacI和Z基因发生了融合。分离出了一个包含融合位点的77个残基的溴化氰肽段。使用针对β-半乳糖苷酶的CNBr2（残基3 - 92）制备的抗体进行放射免疫测定，以监测其纯化过程。通过胰蛋白酶肽段分析和自动测序仪分析确定了连接肽的序列。连接位点是从乳糖阻遏物的第355位残基到β-半乳糖苷酶的第24位残基（或从356到25），这表明乳糖阻遏物羧基末端的最后4个残基和β-半乳糖苷酶氨基末端的前23个残基对于这两种蛋白质的活性并非必需。由于乳糖阻遏物的第356位残基和β-半乳糖苷酶的第24位残基都是亮氨酸残基，所以融合的确切位点尚不清楚。对缺失两端点周围核苷酸序列的检查揭示，在一段11个碱基对的区域中有9个相同碱基的同源性。
