Heiskanen Tuomas, Li Xiao-Dong, Hepojoki Jussi, Gustafsson Elisabeth, Lundkvist Ake, Vaheri Antti, Lankinen Hilkka
Department of Virology, Haartman Institute, P.O. Box 21, FIN-00014 University of Helsinki, Finland.
Protein Eng. 2003 Jun;16(6):443-50. doi: 10.1093/protein/gzg058.
We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVR recognizing the Puumala virus (PUUV) G2-glycoprotein-specific neutralizing monoclonal antibody (MAb) 1C9 with Kd of 2.85 x 10(-8) from a random peptide library X2CX14CX2 expressed on the pIII protein of the filamentous phage fd-tet. We have now created a second-generation phage-displayed peptide library in which each amino acid of the peptide was mutated randomly to another with a certain probability. Peptides were selected for higher affinity for MAb 1C9 and for a common binding motif for MAb 4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein. The resulting peptides were synthesized as spots on cellulose membrane. Amino acid changes which improved the reactivity of the peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCAL with Kd of 1.49 x 10(-9) in biosensor measurements. Our results show that the binding properties of peptides, the affinity and the specificity can be improved and the binding specificity determining amino acids and structural factors can be analyzed by combining binding assays with synthetic peptides on membrane with the use of second-generation phage display libraries.
我们之前从在丝状噬菌体fd-tet的pIII蛋白上表达的随机肽库X2CX14CX2中筛选出了一段肽插入序列FPCDRLSGYWERGIPSPCVR，它能识别普马拉病毒（PUUV）G2糖蛋白特异性中和单克隆抗体（MAb）1C9，解离常数（Kd）为2.85×10⁻⁸。我们现在构建了第二代噬菌体展示肽库，其中肽段的每个氨基酸都以一定概率随机突变为另一种氨基酸。筛选出对MAb 1C9具有更高亲和力以及对MAb 4G2具有共同结合基序的肽段，MAb 4G2在G2糖蛋白中与MAb 1C9具有重叠表位。将所得肽段作为斑点合成在纤维素膜上。在生物传感器测量中，将提高肽段与MAb 1C9反应性的氨基酸变化组合到肽ATCDKLFGYYERGIPLPCAL中，其Kd为1.49×10⁻⁹。我们的结果表明，通过将膜上合成肽的结合测定与第二代噬菌体展示库相结合，可以改善肽段的结合特性、亲和力和特异性，并分析决定结合特异性的氨基酸和结构因素。
