Phage Display Evolution of a Peptide Substrate for Yeast Biotin Ligase and Application to Two-Color Quantum Dot Labeling of Cell Surface Proteins

Abstract

Site-specific protein labeling with Escherichia coli biotin ligase (BirA) has been used to introduce fluorophores, quantum dots (QDs), and photocross-linkers onto recombinant proteins fused to a 15-amino acid acceptor peptide (AP) substrate for BirA and expressed on the surface of living mammalian cells. Here, we used phage display to engineer a new and orthogonal biotin ligase-AP pair for site-specific protein labeling. Yeast biotin ligase (yBL) does not recognize the AP, but we discovered a new 15-amino acid substrate for yBL called the yeast acceptor peptide (yAP), using two generations of phage display selection from 15-mer peptide libraries. The yAP is not recognized by BirA, and thus, we were able to specifically label AP and yAP fusion proteins coexpressed in the same cell with differently colored QDs. We fused the yAP to a variety of recombinant proteins and demonstrated biotinylation by yBL at the N-terminus, C-terminus, and within a flexible internal region. yBL is extremely sequence-specific, as endogenous proteins on the surface of yeast and HeLa cells are not biotinylated. This new methodology expands the scope of biotin ligase labeling to twocolor imaging and yeast-based applications.

Nanoscale bacteriophage biosensors beyond phage display

“…[22][23][24][25][26][27][28][29] The most common form of filamentous bacteriophagebased biosensor is engineered by conjugating targeting moieties and fluorescent molecules onto the capsid surface, ultimately for the detection of target analyte and for the transduction of signal via fluorescence. 30,31 For example, Li et al 9 constructed a bacteriophage capable of targeting a HeLa contaminant KB cell line and emitting fluorescence upon target binding ( Figure 4A) Another common type of genetically-engineered filamentous bacteriophage-based sensor involves the conjugation of nanoparticles to the capsid, in which the unique properties of each type of nanoparticle is exploited in an application-specific manner. [33][34][35] In one instance, Lee et al 10 engineered a bacteriophage with a peptide library against a specific antigen on the p3 subunit and gold (Au) nanoparticles on its p8 subunit.…”

Phage display and molecular imaging: expanding fields of vision in living subjects

“…In one case, phage display was used to evolve a new peptide substrate for yeast biotin ligase, which upon genetic fusion to a protein of interest enables site-specific labeling with imaging probes conjugated to streptavidin (Chen et al 2007). Selections were performed by incubating phage peptide libraries with biotin and yeast biotin ligase, followed by recovery of biotinylated phage clones with streptavidin-coated beads.…”

Rapid isolation and single-molecule analysis of ribonucleoproteins from cell lysate by SNAP-SiMPull

“…Based on this precedent, we created a derivative of the SNAP tag that could be biotinylated in vivo. While several options exist for biotinylation of proteins (Cronan 1990;Chen et al 2007), we chose to utilize the well-characterized biotin acceptor peptide (AP) and Escherichia coli biotin ligase, BirA. BirA catalyzes robust biotinylation of the diminutive 15 amino acid AP when the enzyme is coexpressed in the cell (Schatz 1993;Beckett et al 1999;van Werven and Timmers 2006).…”

“…BirA catalyzes robust biotinylation of the diminutive 15 amino acid AP when the enzyme is coexpressed in the cell (Schatz 1993;Beckett et al 1999;van Werven and Timmers 2006). We predicted that fusion of the AP to the SNAP protein would permit biotinylation of SNAP AP -tagged proteins in the presence of BirA (Chen et al 2007). The SNAP APtagged proteins could then be subsequently labeled with fluorophores, immobilized on a surface, and imaged (Fig.…”