Genetic construction of a phosphorylation site in ricin A chain: specific radiolabeling of recombinant proteins for localization and degradation studies.
Genetic construction of a phosphorylation site in ricin A chain: specific radiolabeling of recombinant proteins for localization and degradation studies.
复制标题
蓖麻毒素 A 链磷酸化位点的遗传构建：重组蛋白的特异性放射性标记用于定位和降解研究。
DOI：
10.1006/bbrc.1995.1654
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Ramakrishnan,S
中科院分区：
文献类型：
--
作者：
Fryxell,D;Li,BY;Mohanraj,D;Johnson,B;Ramakrishnan,S
Ricin A chain was modified by the addition of the heptapeptide LRRASLG (Kemptide) and a histidine rag for bacterial expression. The mutagenized toxin was purified by nickel column and could be phosphorylated in vitro by protein kinase A as demonstrated by labeling with [γ-32P] ATP. Kemptide-A chain could be labeled even after reassociation with ricin B chain or disulfide linkage to antibody to form an immunotoxin. The32P label in all cases was associated only with the A chain; ricin B chain and antibody were not kinase substrates alone or after conjugation. Kemptide-immunotoxin was tested in cytotoxicity assays and used to monitor internalization of the toxin moiety after [32P] phosphorylation.
