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, 128 (14), 4542-3

Transglutaminase-catalyzed Site-Specific Conjugation of Small-Molecule Probes to Proteins in Vitro and on the Surface of Living Cells

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Transglutaminase-catalyzed Site-Specific Conjugation of Small-Molecule Probes to Proteins in Vitro and on the Surface of Living Cells

Chi-Wang Lin et al. J Am Chem Soc.

Abstract

Site-specific protein labeling methods allow cell biologists to access the vast array of existing chemical probes for the study of specific proteins of interest in the live cell context. Here we describe the use of the transglutaminase enzyme from guinea pig liver (gpTGase), whose natural function is to cross-link glutamine and lysine side chains, to covalently conjugate various small-molecule probes to recombinant proteins fused to a 6- or 7-amino acid transglutaminase recognition sequence, called a Q-tag. We demonstrate labeling of Q-tag fusion proteins both in vitro and on the surface of living mammalian cells with biotin, fluorophores, and a benzophenone photoaffinity probe. To illustrate the utility of this labeling, we tagged the NF-kappaB p50 transcription factor with benzophenone, cross-linked with UV light, and observed increased levels of p50 homodimerization in the presence of DNA and the binding protein myotrophin.

Figures

Figure 1
Figure 1
Transglutaminase-catalyzed ligation of cadaverine-functionalized probes (green circle) to Q-tag-fused recombinant cell surface proteins. TGase ligates the glutamine sidechain of the Q-tag (blue) to the amine probe, giving an amide bond and releasing ammonia. In this study, three Q-tag peptide substrates were used: Q1 (PNPQLPF), Q2 (PKPQQFM), and Q3 (GQQQLG).
Figure 2
Figure 2
TGase-catalyzed labeling of Q-tagged cell surface protein. The domain structure of Q2-CFP-TM is shown, from N- to C-terminus. TM is the transmembrane domain of the PDGF receptor, which targets Q2-CFP to the cell surface. HeLa cells expressing Q2-CFP-TM were labeled with Alexa 568 cadaverine by incubating with the probe, gpTGase, and CaCl2 for 25 min at 4 °C. The top row shows the CFP images superimposed on the DIC images. The bottom row shows the Alexa 568 fluorescence. Negative controls are shown with the alanine mutant Q2(Ala)-CFP-TM and with gpTGase enzyme left out.
Figure 3
Figure 3
Labeling p50-Q2 with benzophenone and photoinduced cross-linking. p50-Q2 protein was labeled with benzophenone spermine using gpTGase and CaCl2 for 1.5 hr at 37 °C. After Ni-NTA purification to remove excess benzophenone, the protein was irradiated with UV light for 7 min at 4 °C, in the presence or absence of DNA (0.18 μM) or myotrophin (360 μM; 90 equivalents over p50). The products were separated on 10 % SDS-PAGE and blotted with anti-p50 antibody. Formation of p50 dimer (MW 78.5 kD) is seen in lanes 8–10, but not in others where the alanine mutant was used, gpTGase was omitted, or the samples were not UV-irradiated.

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