Directing sequence-specific proteolysis to new targets - The influence of loop size and targets sequence on selective proteolysis by tissue-type plasminogen activator
Directing sequence-specific proteolysis to new targets - The influence of loop size and targets sequence on selective proteolysis by tissue-type plasminogen activator
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DOI：
10.1074/jbc.273.8.4323
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发表时间：
1998-02-20
影响因子：
4.8
通讯作者：
Corey, DR
中科院分区：
文献类型：
--
作者：
Coombs, GS;Bergstrom, RC;Corey, DR
我们之前使用底物噬菌体展示来鉴定可被组织型纤溶酶原激活剂 (t-PA) 或尿激酶型纤溶酶原激活剂 (u-PA) 有效选择性切割的肽序列，我们证明该信息可用于将选择性蛋白水解定向到新的蛋白质靶标，当在肽的背景下时，对 t-PA 或 u-PA 选择性切割不稳定的序列被引入 43-52葡萄球菌核酸酶（或 Omega）环，t-PA 和 u-PA 都能水解插入靶序列处的工程蛋白，并且相对于 15 个残基肽内类似序列的裂解值，蛋白质裂解的 K-m 值降低高达 200 倍。靶序列周围环大小的变化对 t-PA 效率的影响比胰蛋白酶强约 5 倍，这表明裂解t-PA 的切割更依赖于靶位点的迁移率，t-PA 和 u-PA 对蛋白质的切割具有序列选择性，对于小肽底物内已知为 u-PA 选择性的序列的切割，u-PA 的活性比 t-PA 高 47 倍，而 t-PA 对 t-PA 选择性序列的活性高 230 倍。
We have previously used substrate phage display to identify peptide sequences that are efficiently and selectively cleaved by tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA), We demonstrate that this information can be used to direct selective proteolysis to new protein targets, Sequences that were labile to selective cleavage by t-PA or u-PA when in the context of a peptide were introduced into the 43-52 (or Omega) loop of staphylococcal nuclease, Both t-PA and u-PA hydrolyze the engineered proteins at the inserted target sequences, and K-m values for protein cleavage were reduced up to 200-fold relative to values for cleavage of analogous sequences within 15 residue peptides, Variation of loop size surrounding a target sequence affects the efficiency of t-PA approximately 5-fold more strongly than that of trypsin, suggesting that cleavage by t-PA is more dependent on target site mobility, Cleavage of proteins by t-PA and u-PA is sequence selective, u-PA is 47-fold more active than t-PA for cleavage of a sequence known to be u-PA selective within small peptide substrates, whereas t-PA is 230-fold more active toward a t-PA-selective sequence.