Protein Chemical Modification Inside Living Cells Using Split Inteins

Abstract

Methods to visualize, track, measure, and perturb or activate proteins in living cells are central to biomedical efforts to characterize and understand the spatial and temporal underpinnings of life inside cells. Although fluorescent proteins have proven to be extremely useful for in vivo studies of protein function, their utility is inherently limited because their spectral and structural characteristics are interdependent. These limitations have spurred the creation of alternative approaches for the chemical labeling of proteins. We describe in this protocol the use of fluorescence resonance emission transfer (FRET)-quenched DnaE split-inteins for the site-specific labeling and concomitant fluorescence activation of proteins in living cells. We have successfully employed this approach for the site-specific in-cell labeling of the DNA binding domain (DBD) of the transcription factor YY1 using several human cell lines. Moreover, we have shown that this approach can be also used for modifying proteins in order to control their cellular localization and potentially alter their biological activity.

Keywords: Fluorescence; Npu intein; Protein labeling; Protein trans-splicing; Split-intein.

Figure 1

A. Site-specific labeling and fluorescence activation of a protein of interest (POI) by FRET-quenched protein trans-splicing. Key to this approach is the introduction of fluorescence quencher (Q) into the I_C polypeptide, which blocks the fluorescence signal of the fluorophore (F) located at the C-terminus of the I_C polypeptide before protein trans-splicing happens. When protein trans-splicing occurs the fluorophore is covalently attached to the C-terminus of the POI triggering its fluorescence. The use of this approach for in-cell modification and fluorescence tagging of proteins minimizes the fluorescence background from the unreacted I_C polypeptide thus facilitating the optical tracking of the labeled protein inside the cell. B. Scheme showing the approach used for in-cell labeling of a POI with a fluorophore inside a live cell using protein trans-splicing (figure modified from [13]).

Figure 2

Design of FRET-quenched DnaE split inteins. A. Multiple sequence alignment of the DnaE I_C for different species indicating the positions used for the introduction of the quencher group in the I_C polypeptide. Multiple sequence alignment was performed using T-Coffee and visualized using Jalview [26]. Molecular representations of the DnaE inteins were generated using the PyMol software package. B. Crystal structure of the Npu DnaE intein in the pre-spliced state (PDB code: 2KEQ) [27]. DnaE I_C and I_N are shown in red and blue respectively. The structural secondary elements are also shown. The position used to place the quencher and fluorophore groups at the I_C and C-extein, as well as the distances are indicated (figure modified from [13]).

Figure 3

SDS-PAGE analysis of the in vitro protein trans-splicing/labeling reaction between FRET-quenched DnaE I_C (Table 1) and YY1-I_N. Protein detection was performed by silver staining (top) and epifluorescence (bottom). TS = trans-splicing.

Figure 4

Expression of protein YY1-I_N in U2OS (and HeLa) cells. Cells were collected after 24 h post-transfection, lysed and the soluble fraction analyzed and quantified by western blot using an anti-His antibody.

Figure 5

In-cell site-specific labeling of YY1 DBD with a nuclear localization signal and concomitant fluorescence activation using protein trans-splicing. A. U2OS (and HeLa) cells were first transiently transfected with a plasmid encoding YY1-I_N and with DnaE I_C polypeptides as described. Cells were then extensively washed and examined by fluorescence microscopy. B. Magnification of cells after 18 h of incubation showing the migration of the labeled-YY1 to the nuclear compartment. C. Quantification of labeling yield for in-cell trans-splicing reaction. Identification of labeled YY1 DBD protein and quantification of in-cell trans-splicing yield was performed by western blot (right panel) and epifluorescence (left panel), respectively. Bar represents 25 μm in panels A and B. TS = trans-splicing.