Berg A P, Fowler A V, Zabin I
J Bacteriol. 1970 Feb;101(2):438-43. doi: 10.1128/jb.101.2.438-443.1970.
A prematurely terminated polypeptide chain was purified to homogeneity from an Escherichia coli amber mutant strain containing the site of the mutation in the beta-galactosidase structural gene. The polypeptide was highly active against anti-beta-galactosidase, and had an amino acid composition similar to but not identical to that of beta-galactosidase. The molecular weight of the reduced, carboxymethylated chain in 6 m guanidine hydrochloride was found to be 89,000, in excellent agreement with the size predicted from the position of the mutation. This result adds further support to the conclusion that the gene specifies the structure of a single polypeptide chain. Antisera were prepared against partially purified preparations of this polypeptide and a similar one, of molecular weight about 100,000, produced by another amber mutant. These sera had lower titers towards beta-galactosidase than anti-beta-galactosidase. In the double-diffusion test, they reacted towards extracts of nonsense and deletion mutant strains in a pattern similar to that previously observed with anti-beta-galactosidase. A sensitive immunological test for cross-reacting protein was devised based on the inhibition by beta-galactosidase of the reaction between such protein and antibodies prepared against incomplete chains.
从β-半乳糖苷酶结构基因中含有突变位点的大肠杆菌琥珀突变株中纯化出一条提前终止的多肽链，使其达到同质。该多肽对抗β-半乳糖苷酶具有高活性，其氨基酸组成与β-半乳糖苷酶相似但不相同。在6M盐酸胍中还原的、羧甲基化的链的分子量为89,000，与根据突变位置预测的大小非常一致。这一结果进一步支持了基因指定单一多肽链结构的结论。针对该多肽的部分纯化制剂以及另一个琥珀突变体产生的分子量约为100,000的类似多肽制备了抗血清。这些血清对β-半乳糖苷酶的效价低于抗β-半乳糖苷酶。在双向扩散试验中，它们与无义突变和缺失突变株的提取物反应的模式与先前用抗β-半乳糖苷酶观察到的模式相似。基于β-半乳糖苷酶对这种蛋白质与针对不完全链制备的抗体之间反应的抑制作用，设计了一种用于交叉反应蛋白的灵敏免疫检测方法。
