B

c

Fig. 4.4.3. Covalent labeling of nuclear targeted W160hAGT-NLS3 in AGT-deficient CHO cells. Confocal micrographs A-C show overlays of transmission and fluorescence channels (exc 488 nm). The size bar in A-C corresponds to 10 mm. Confocal micrographs (A-C) illustrate the time course of the labeling of transiently expressed W160hAGT-NLS3 with BGAF in AGT-deficient CHO cells. (A) AGT-deficient CHO

cell transiently expressing W160hAGT-NLS3 during incubation with BGAF (5 |im). (B) Same cell as in (A) after 5 min incubation with BGAF (5 |im) and three washes with PBS. (C) Same cell as in (B) after additional 25 min incubation in PBS, illustrating the specific labeling of hAGT-NLS3 in the nucleus of a transiently transfected CHO cell.

Fig. 4.4.3. Covalent labeling of nuclear targeted W160hAGT-NLS3 in AGT-deficient CHO cells. Confocal micrographs A-C show overlays of transmission and fluorescence channels (exc 488 nm). The size bar in A-C corresponds to 10 mm. Confocal micrographs (A-C) illustrate the time course of the labeling of transiently expressed W160hAGT-NLS3 with BGAF in AGT-deficient CHO cells. (A) AGT-deficient CHO

cell transiently expressing W160hAGT-NLS3 during incubation with BGAF (5 |im). (B) Same cell as in (A) after 5 min incubation with BGAF (5 |im) and three washes with PBS. (C) Same cell as in (B) after additional 25 min incubation in PBS, illustrating the specific labeling of hAGT-NLS3 in the nucleus of a transiently transfected CHO cell.

cause of covalent labeling of W160hAGT-NLS3. First, in AGT-deficient CHO cells not transfected with the gene of the hAGT fusion protein no fluorescence labeling of the nucleus was observed. Second, incubating CHO cells transiently expressing W160hAGT-NLS3 with BG (10 mm) before incubation with BGAF to inactivate the hAGT fusion protein prevented the fluorescence labeling of the nucleus. The data thus show that hAGT can be covalently and specifically labeled in mammalian cell cultures. The concentration of fluorescence-labeled W160hAGT-NLS3 in the nucleus was estimated to be 2 ^m. By comparison, the concentration of the W160hAGT-ECFP-NLS3 fusion protein in the nucleus after 24 h of transient expression was estimated by fluorescence of ECFP to be 3 mm, demonstrating that the in vivo concentration of the labeled hAGT fusion protein approaches that of the corresponding ECFP fusion protein.

To improve the speed and the efficiency of the in-vivo labeling and to obtain novel insights into the structure-function relationship of the protein, we also generated hAGT mutants with increased activity against BG derivatives. In these experiments, we chose to submit hAGT to directed evolution using phage display [16, 17] (Box 20). In the following phage display experiments hAGT was displayed as a fusion protein with the phage capsid protein pill. Selections were based on incubating phages with BGDG and isolating those phages covalently labeled with digoxigenin, by use of immobilized anti-digoxigenin antibodies. To select for hAGT mutants with increased activity against BGDG, we randomized four amino acids that were in the proximity of either the benzyl ring (Pro140, Gly159, Gly160), or could make contact with the purine (Asn157). Using phage display, hAGT mutants were selected which had up to fifteenfold increased activity against BGBT than wild-type, the most active mutant with the sequence Pro140, Gly157, Glu159, Ala160 (PGEA). To demonstrate that these mutants can improve the efficiency of our in-vivo labeling technique, the mutant PGEA was transiently expressed as a PGEA-NLS3 fusion protein in AGT-deficient CHO cells and its labeling with BGFL investigated by confocal fluorescence microscopy. In these experiments, the mutant PGEA led to a twofold higher fluorescence signal in the nucleus of the cell than the previously used mutant W160hAGT.

Besides its unusual mechanism, hAGT has a variety of properties that make it a suitable protein for specific and covalent labeling of fusion proteins in vivo. Most importantly, it has high reactivity against a substrate which is otherwise chemically inert and which can be derivatized by a wide variety of labels such as dyes, cross-linkers, or affinity tags without significantly affecting the rate of the reaction of hAGT with the substrate. None of the substrates tested so far had chemical toxicity during the in-vivo labeling and, because of the irreversibility of the labeling, excess substrate can be easily washed away. hAGT is, furthermore, a monomer of 207 residues, thereby reducing the likelihood that its fusion to other proteins will affect their oligomeric state, and the protein of interest can be fused either to the N or the C terminus of hAGT without affecting its reactivity. Experiments in mammalian cells should be performed in AGT-deficient cell lines, preventing the labeling of the endogenous AGT. For example, incubating HEK293 cells with BGAF leads to detectable fluorescence labeling of the endogenous AGT in the nucleus of the cell. The efficiency of the in vivo labeling for a given substrate will depend primarily on its cell permeability. For most applications, however, quantitative labeling of the protein of interest is not mandatory.

In conclusion, we have developed a general method enabling covalent and specific labeling of fusion proteins in vivo. Its applicability in different organisms and its independence of the nature of the label should make this method an important tool for functional studies of proteins in the living cell. AGT fusion proteins should, furthermore, become a powerful tool for all those in-vitro applications where the protein of interest must be either specifically labeled or immobilized.

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