Site-specific Labeling of a Protein Lysine Residue By Novel Kinetic Labeling Combinatorial Libraries.
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.
Advanced Proteome Therapeutics Inc., 650 Albany Street, Suite 113, Boston, MA 02118, United States ; RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.).
Allen
A
Krantz
RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.).
Arthur M
AM
Hanel
Advanced Proteome Therapeutics Inc., 650 Albany Street, Suite 113, Boston, MA 02118, United States ; Current address: EMD Millipore, 17 Cherry Hill Drive, Danvers, Massachusetts, 01923, United States.
Ivona
I
Strug
Advanced Proteome Therapeutics Inc., 650 Albany Street, Suite 113, Boston, MA 02118, United States.
Andrzej
A
Wilczynski
Bruker Daltonics Inc., 40 Manning Road, Billerica, MA 01821, United States.
Jeremy J
JJ
Wolff
RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.).
Wolin
W
Huang
RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.).
Linda H
LH
Huang
RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.) ; Current address: Thermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134, United States.
Tina
T
Settineri
RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.).
Darren L
DL
Holmes
RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.).
Margaret C
MC
Hardy
RedCell Inc., 270-B Littlefield Avenue, South San Francisco, CA, 94080, United States (Renamed ConjuChem LLC. Current address: 11755 Wilshire Blvd, Suite 2000, Los Angeles, CA 90025.) ; Current address: Optivia Biotechnology Inc., 115 Constitution Drive, Suite 7, Menlo Park, CA 94025, United States.
Dominique P
DP
Bridon
Affinity labels; Combinatorial libraries; Kinetic labeling libraries; Lysine labeling; Peptide libraries; Site-specific protein modification