Cheadle C, Ivashchenko Y, South V, Searfoss G H, French S, Howk R, Ricca G A, Jaye M
Department of Molecular Biology, Rhône-Poulenc Rorer Central Research, Collegeville, Pennsylvania 19426.
J Biol Chem. 1994 Sep 30;269(39):24034-9.
A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.
在丝状噬菌体fuse5中构建了一个噬菌体展示文库。该文库通过将编码15个可变氨基酸的简并寡核苷酸插入噬菌体基因III蛋白的NH2末端区域而制成。这个包含超过10^7种不同噬菌体的文库，用含有Src同源3（Src SH3）结构域和蛋白激酶A磷酸化位点的谷胱甘肽S-转移酶（GST）融合蛋白（GST/PKA/Src SH3）进行筛选。经过四轮富集和扩增后，分离出了一系列富含脯氨酸的序列。含有这些序列的噬菌体被证明能特异性结合GST/PKA/Src SH3蛋白，而不与仅GST/PKA结合。对不同噬菌体克隆推导的氨基酸序列进行比较，揭示了一个共有序列RPLPXXP，它符合在使用32P标记的Src SH3蛋白片段作为探针的λ-lox小鼠胚胎cDNA文库的亲和筛选过程中独立鉴定出的Src SH3结构域结合基序（Y. Ivashchenko，正在准备的手稿）。基于7个氨基酸的SH3结合结构域核心基序的肽对Src和Fyn的SH3结构域都显示出强结合，但不能结合p21 Ras - GTP酶激活蛋白（Ras - GAP）和其他蛋白的SH3结构域。我们预计，对噬菌体展示文库的进一步筛选将成为快速鉴定其他SH3结构域结合序列的有用工具，也将有助于确定定义含有SH3结构域的不同蛋白质与含有SH3结合基序的蛋白质之间相互作用特异性的基本核心基序。
