Site-specific labeling of a protein lysine residue by novel kinetic labeling combinatorial libraries

Publication date: March 2014 Source:Computational and Structural Biotechnology Journal, Volume 9, Issue 15 Author(s): Allen Krantz , Arthur M Hanel , Ivona Strug , Andrzej Wilczynski , Jeremy J Wolff , Wolin Huang , Linda H Huang , Tina Settineri , Darren L Holmes , Margaret C Hardy , Dominique P Bridon The first example of a kinetic labeling library designed to enable the discovery of affinity labels is presented. Each library component (1) consists of a variable peptidyl component linked to a biotinyl moiety by a 4-mercaptobenzoyl linker in thioester format. We demonstrate that an affinity label can be uncovered by measuring reaction rates between library pools and the protein target, human serum albumin (HSA) and identifying significant outliers. By choosing peptide functionality compatible with a potentially reactive thioester labeling entity, libraries can be screened in pools. It is noteworthy that a limited subset of amino acids (R, S, E, F, Y, l, M, W, and Q) that compose the affinity moiety is sufficient to produce rate variances that guide the discovery process. After two rounds of deconvolution, J-FLYEE-NH2 (7-E) emerges as a bona fide affinity label of HSA. Unlike known affinity labels, the affinity moiety is not retained in the protein product, but is extruded upon acylation of the protein. This feature affords a method of introducing various payloads, without extraneous elements, onto protein frameworks.
Source: Computational and Structural Biotechnology Journal - Category: Biotechnology Source Type: research