A solid-phase screen for protein kinase substrate selectivity.
A solid-phase screen for protein kinase substrate selectivity.
复制标题
蛋白激酶底物选择性的固相筛选。
DOI：
10.1016/0003-2697(92)90313-v
复制
发表时间：
1992
影响因子：
2.9
通讯作者：
Kuret,J
中科院分区：
文献类型：
--
作者：
Carmel,G;Kuret,J
盒式诱变用于合成具有独特磷酸化位点的大肠杆菌表达文库。该基因盒编码一个丝氨酸残基，由Ala、Arg、Gln、Glu、Gly和Pro残基组成的7个氨基酸残基（共67个氨基酸残基）。将该表达盒插入到合适的载体蛋白基因中，并在E. coli与T7表达系统，并将所得文库在硝酸纤维素滤膜上进行固相蛋白磷酸化测定。当用TPK 1 Δ（酿酒酵母cAMP依赖性蛋白激酶的修饰催化亚基）筛选文库时，鉴定了表达该激酶底物的单个菌落。通过对阳性克隆的盒区进行DNA测序，推断出TPK 1 Δ的共有识别序列，并发现其与哺乳动物同源物（Arg-Arg-Xaa-Ser）的公认底物选择性一致。由于大量的克隆可以被快速测序，并确定了组成识别位点的不变残基的位置，这种方法可能是有用的蛋白激酶底物选择性的一般屏幕。
Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites. The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 67≈ 2.8 × 105sequences). The cassette was inserted into the gene of a suitable carrier protein and expressed in E. coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters. When the library was screened with TPK1Δ, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified. By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1Δ was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser). Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity.