Krieger D E, Erickson B W, Merrifield R B
Proc Natl Acad Sci U S A. 1976 Sep;73(9):3160-4. doi: 10.1073/pnas.73.9.3160.
A general strategy and a specific tactic for affinity purification of polypeptides synthesized on solid supports are desbribed and demonstrated. The desired peptide chains were distinguished from terminated peptide chains before removal from the support by attachment of an affinity reagnet (cysteinyl-methionine) bearing an affinity group (thiol) and a binding group (carboxylic acid). After cleavage from the synthetic support, the affinity-labeled peptides (Cys-Met-peptides) were bound to an affinity receptor (organomercurial-agarose) and thus separated from terminated peptides and all other peptides lacking the affinity group. The desired synthetic peptide was obtained by separation of the affinity reagent (loss of Cys-Met by cyanogen bromide cleavage). This general affinity purification strategy is independent of the length or amino acid sequence of the desired peptide. After assembly of ribonuclease-(111-124)-tetradecapeptide, using radiolabeled acetic anhudride for termination of uncoupled in termediates, essentially all (greater than 98.5%) of the acetylated delection peptides were removed by employing the organomercurial Cys-Met tactic. Similarly, the purity of crude synthetic histone H4-(1-37)-heptatriacontapeptide was increased six-fold by using this tactic to remove terminated peptides. A related dimeric Cys-Met tactic is outlined for affinity purification of peptides containing internal cysteine and methionine residues.
本文描述并展示了一种用于亲和纯化在固体支持物上合成的多肽的通用策略和具体方法。在从支持物上移除之前，通过连接带有亲和基团（巯基）和结合基团（羧酸）的亲和试剂（半胱氨酰 - 甲硫氨酸），将所需的肽链与终止的肽链区分开来。从合成支持物上切割后，亲和标记的肽（半胱氨酸 - 甲硫氨酸肽）与亲和受体（有机汞琼脂糖）结合，从而与终止的肽和所有其他缺乏亲和基团的肽分离。通过分离亲和试剂（用溴化氰切割去除半胱氨酸 - 甲硫氨酸）获得所需的合成肽。这种通用的亲和纯化策略与所需肽的长度或氨基酸序列无关。在组装核糖核酸酶 -（111 - 124）- 十四肽后，使用放射性标记的乙酸酐终止未偶联的中间体，通过采用有机汞半胱氨酸 - 甲硫氨酸方法，基本上所有（大于98.5%）的乙酰化缺失肽都被去除。同样，通过使用这种方法去除终止的肽，粗合成组蛋白H4 -（1 - 37）- 三十七肽的纯度提高了六倍。本文还概述了一种相关的二聚体半胱氨酸 - 甲硫氨酸方法，用于亲和纯化含有内部半胱氨酸和甲硫氨酸残基的肽。
