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 non-specificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. 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. First, a genetic strategy introduced a short charge neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7+) reduced or eliminated non-specific binding to the problematic high pI proteins. In the second, chemical approach, oligolysine wrappers for phage and total RNA blocked non-specific interactions. For phage display applications, the peptides Lysn (where n = 16 to 24) emerged as optimal for wrapping the phage. Lys8, however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked non-specific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.
The cover picture shows a section of the M13 filamentous bacteriophage, widely used for the display and manipulation of proteins in both academic and industrial laboratories. The negatively charged outer surface of conventional phage (red coat proteins at the bottom third of the phage) results in nonspecific binding to high-pI target proteins such as colicin E9 DNase (green) and lysozyme (purple). All too often such nonspecific binding results in failed screens, assays and selections. As shown on p. 2846 ff., G. Weiss et al. solve this problem by inserting a genetically encoded positively charged peptide into the phage coat (blue coat proteins in the middle of the phage). A chemical approach with oligolysine wrappers can also mask the negatively charged surface (top third of the phage) to allow experiments with previously inaccessible target proteins. Cover art by Denise Der and Jorge Lamboy from a design by Gregory Weiss.
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