Site-specific protein conjugates incorporating Para-Azido-L-Phenylalanine for cellular and in vivo imaging

Abstract

This work features the use of amber suppression-mediated unnatural amino acid (UAA) incorporation into proteins for various imaging purposes. The site-specific incorporation of the UAA, p-azido-L-phenylalanine (pAzF), provides an azide handle that can be used to complete the strain promoted azide-alkyne click cycloaddition (SPAAC) reaction to introduce an imaging modality such as a fluorophore or a positron emission tomography (PET) tracer on the protein of interest (POI). Such methodology can be pursued directly in mammalian cell lines or on proteins expressed in vitro, thereby conferring a homogeneous pool of protein conjugates. A general procedure for UAA incorporation to use with a site-specific protein labeling method is provided allowing for in vitro and in vivo imaging applications based on the representative proteins PTEN and PD-L1. This approach would help elucidate the cellular or in vivo biological activities of the POI.

Keywords: Fluorescent imaging; Site-specific protein labeling; positron emission tomography (PET); unnatural amino acid (UAA).

Figure 1.

A general scheme to demonstrate the mechanism of unnatural amino acid (UAA) incorporation through amber-mediated suppression. Figure was adapted with permission from Wang,Q., Parrish,A.R., & Wang,L. (2009). Expanding the genetic code for biological studies. Chemical Biology, 16(3), 323–336. Copyright (2009) Elsevier Ltd.

Figure 2:

Site-specific amber suppression on the protein of interest to incorporate p-azido-lphenylalanine (pAzF), followed by SPAAC reaction with the bicyclo[6.1.0]non-4-yne (BCN) moiety of the imaging modality of choice. For PET imaging, 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) was employed here. For fluorescent imaging, tetramethylrhodamine (TAMRA) was used for live-cell applications.

Figure 3.

(A) PTEN protein structure (PDB 1D5R) created via Pymol, with the site of UAA indicated by X in the sequence. (B) The Plasmid vector maps for PTEN and the vector for tRNA/synthetase. (C) Live-cell imaging results with the first row showing cells having received pAzF treatment, with TAMRA fluorescence. The second row was a vehicle control without pAzF treatment, which displayed no discernible fluorescence as the lack of pAzF prevented amber suppression. The transmission channel confirms the presence of fluorescence within the HEK293 cells, and the blue channel indicates Hoechst nuclear staining. (D) The SDS-PAGE based in-gel fluorescence detection of TAMRA labeling and the western blot detection of PTEN protein. The lanes are marked as: 1. Ladder; 2. Untransfected HEK293 cells; 3. Transfected HEK293 cells with the treatment of pAzF; 4. Vehicle control using transfected HEK293 cells but without pAzF treatment.

Figure 4.

(A) αPD-L1 protein structure with the potential mutation sites highlighted for in silico simulation. (B) In vitro PAGE gel characterization and MS characterization of protein before and after conjugation. (C) In vitro ELISA to confirm affinity was not compromised by the mutation and the conjugation. (D) In vivo PET imaging result. The white arrow is pointed to the spleen and the yellow arrow points to brown adipose tissue. Figure was adapted with permission from Wissler,H.L., Ehlerding,E.B., Lyu,Z., Zhao,Y., Zhang,S., Eshraghi,A., et al. (2019). Site-specific immuno-PET tracer to image PD-L1, Molecular Pharmaceutics, 16, 2028—2036. Copyright (2019) American Chemical Society