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Targeted in vitro evolution of protein ligands.

DE2001767437

Publication Date	2000
Personal Author	Lehnert, B.; Allen, D.
Page Count	8
Abstract	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).
Keywords	In vitro Proteins Ligands RNA DNA Peptides
Source Agency	Technical Information Center Oak Ridge Tennessee
NTIS Subject Category	57F - Cytology, Genetics, & Molecular Biology 57B - Biochemistry
Corporate Authors	Los Alamos National Lab., NM.; Department of Energy, Washington, DC.
Document Type	Technical Report
NTIS Issue Number	200124
Contract Number	W-7405-ENG-36

Targeted in vitro evolution of protein ligands.

Targeted in vitro evolution of protein ligands.
DE2001767437

Publication Date	2000
Personal Author	Lehnert, B.; Allen, D.
Page Count	8
Abstract	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).
Keywords	In vitro Proteins Ligands RNA DNA Peptides
Source Agency	Technical Information Center Oak Ridge Tennessee
NTIS Subject Category	57F - Cytology, Genetics, & Molecular Biology 57B - Biochemistry
Corporate Authors	Los Alamos National Lab., NM.; Department of Energy, Washington, DC.
Document Type	Technical Report
NTIS Issue Number	200124
Contract Number	W-7405-ENG-36