Identification of epitope-like consensus motifs using mRNA display
Identification of epitope-like consensus motifs using mRNA display
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DOI：
10.1002/jmr.567
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发表时间：
2002-05-01
影响因子：
2.7
通讯作者：
Wagner, RW
中科院分区：
文献类型：
--
作者：
Baggio, R;Burgstaller, P;Wagner, RW
体外蛋白质选择的mRNA展示方法是基于嘌呤霉素介导的mRNA与其基因产物之间共价键的形成。该技术可用于鉴定参与大分子识别的肽序列，包括与天然配体表位相同或同源的肽序列。为了证明这种方法，我们确定了胰蛋白酶活性位点和抗c-Myc抗体9 E10识别的肽序列。在这里，我们描述了使用两个不同的二聚体的肽库，一个是基于胰蛋白酶抑制剂EETI-II的限制性文库，其中只有第一个环中的六个残基是随机的（6.4 × 10（7）个可能的序列，文库中的6.0 × 10（11）个序列），另一个是具有27个随机氨基酸的线性肽文库（1.3 × 10（35）个可能的序列，文库中2 × 10（13）个序列）。针对野生型EETI的天然靶标牛胰蛋白酶筛选约束文库，并且针对抗c-myc抗体9 E10筛选线性文库。所选序列的分析揭示了EETI-II第一环的PR（LLV）L和9 E10表位的LISE的最小共有序列。以最高频率选择野生型序列，EETI-II第一环的PRILMR和9 E10表位的QKLISE，并且在每种情况下，从文库中选择完整的野生型表位。版权所有（C）2002约翰威利父子有限公司
The mRNA display approach to in vitro protein selection is based upon the puromycin-mediated formation of a covalent bond between an mRNA and its gene product. This technique can be used to identify peptide sequences involved in macromolecular recognition, including those identical or homologous to natural ligand epitopes. To demonstrate this approach, we determined the peptide sequences recognized by the trypsin active site, and by the anti-c-Myc antibody, 9E10. Here we describe the use of two peptide libraries of different diversities, one a constrained library based on the trypsin inhibitor EETI-II, where only the six residues in the first loop were randomized (6.4 x 10(7) possible sequences, 6.0 x 10(11) sequences in the library), the other a linear-peptide library with 27 randomized amino acids (1.3 x 10(35) possible sequences, 2 x 10(13) sequences in the library). The constrained library was screened against the natural target of wild-type EETI, bovine trypsin, and the linear library was screened against the anti-c-myc antibody, 9E10. The analysis of selected sequences revealed minimal consensus sequences of PR(LLV)L for the first loop of EETI-II and LISE for the 9E10 epitope. The wild-type sequences, PRILMR for the first loop of EETI-II and QKLISE for the 9E10 epitope, were selected with the highest frequency, and in each case the complete wildtype epitope was selected from the library. Copyright (C) 2002 John Wiley Sons, Ltd.