Targeted in vitro evolution of protein ligands.

[摘要] The objective of this project was to create a diverse peptide library by exploiting the MS2 coat protein/RNA operator interaction. In order to accomplish this it was necessary to define the optimal scaffolding for the library in which DNA, coding for the stem loop operator, is fused to tandem coat protein monomers which, when translated, would form a covalent dimer subsequently binding the nascent RNA. Therefore, a randomized library region downstream of the coat protein monomers would also be translated and bound to the coding transcript allowing us to generate a widely diverse library as well as couple sequence to function. As proof of principle to define the scaffolding, we used the c-myc epitope to emulate our N-library region and performed binding and enrichment studies using an anti-myc immunoprecipitation followed by RT PCR. In order to simulate the library environment, which would contain upwards of 10II different peptides, we used a competitor construct, which contained the covalent dimer/operator but lacked the myc epitope. To date, we have successfully competed our target transcript versus competitor at a molar ratio of 1:10(sub 3).

[发布机构] Technical Information Center Oak Ridge Tennessee

[学科分类] 工程和技术（综合）

[关键词] In vitro;Proteins;Ligands;RNA;DNA