Journal
CHEMBIOCHEM
Volume 9, Issue 17, Pages 2846-2852Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.200800366
Keywords
electrostatic interactions; high-pl proteins; lysine; nonspecific binding; phage display
Funding
- National Institutes of Health [1R43A11955-01]
- NIH Minority Fellowship [NIGMS 1R01-GM078528-01]
- National Research Service Award Fellowship
- National Library of Medicine [5T15LM00744]
- AIDS Research and Reference Reagent Program
- Division of AIDS
- NIAID
- National Research Foundation of Korea [과06B1508] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pl proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.
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