Tagging proteins

Ubiquitin tags proteins for protein degradation. The ubiquitination requires three different enzymatic activities, a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme (E2 or Ubc) and a ubiquitin ligase (E3). The action of all three enzymes leads to the establishment of a poly-ubiquitin chain on target proteins which are then recognized and proteolyzed by the 26S proteasome. 

The calculated value for the Hisg-tagged protein is 28,492 Da. N-terminal amino acid sequence analysis (Procise) was carried out on purified recombinant resilin. The following sequence (at 120 pmol yield) was obtained for the first 12 amino acid residues MHHHHHHPEPPV, as expected from DNA sequence analysis. 

Kamoto M, Umezawa N, Kato N, Higuchi T (2009) Turn-on fluorescent probe with visible light excitation for labelling of hexahistidine tagged protein. Bioorg Med Chem Lett 19 2285-2288... 

Yokoe H, Meyer T (1996) Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nat Biotechnol 14 1252-1256... 

Albers, S.V., Jonuscheit, M., Dinkelaker, S. et al. (2006) Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus. Applied and Environmental Microbiology, 72 (1), 102-111. 

Genes of interest can be tagged at either the N or C terminal end. The decision to tag a protein at either the N or the C terminal depends upon the properties of the protein of interest. In our case, all the eukaryotic translation initiation factors were tagged C terminally to allow the endogenous promoter to influence the expression of the tagged protein. 

In addition, Western analysis can be used to verify that the tag has been efficiently added to the protein and that the resultant tagged protein s abundance is not altered by the presence of the tag. Using either an antibody specific to your protein of interested or an anti-GFP antibody (Clontech), a correctly tagged protein should be approximately 29 kDa heavier than the untagged protein (Fig. 3.ID). 

Gautier, I., Tramier, M., Durieux, C., Coppey, J., Pansu, R. B., Nicolas, J. C., Kemnitz, K. and Coppey-Moisan, M. (2001). Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins. Biophys. J. 80, 3000-8. 

Lauf, U., Lopez, P. and Falk, M. M. (2001). Expression of fluorescently tagged connexins A novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498, 11-5. 

Fig. 10.2. FSPIM analysis of the interaction between maize transcriptional coactivators—GCN5 and ADA2—fused to CFP and YFP. GCN5 is a histone acetyltransferase that, in conjunction with adaptor protein ADA2, modulates transcription in diverse eukaryotes by affecting the acetylation status of the core histones in nucleosomes [63]. CFP- and YFP-tagged proteins, expressed in protoplasts, were excited by the 458 nm and the 514 nm laser lines sequentially. CFP fluorescence was selectively detected by an FIFT 458 dichroic mirror and BP 470-500 band pass emission filter while YFP fluorescence was selectively detected by using an HFT 514 dichroic mirror and...

Excellent protocols for transient expression of fluorescently tagged proteins in N. benthamiana leaves using Agrobacterium-mediated infiltration are available [99, 100],... 

The use of pairs of matched spectral variants of GFP, like cyan and yellow or GFP and mRed, to differentially tag proteins and look for interactions, is now in routine use. The approach can be readily applied to homodimerization of molecules that differ only by their living color or epitope tag. For example, homomultimer-ization of a viral coat protein can be observed by imaging a mixture of cyan and yellow tagged homomeric molecules [46], whereas oc-synuclein stacking can be detected by utilizing a-synuclein transcripts that encoded different epitope tags for detection by immunostaining [47],... 

Bhunia, A. K. and Miller, S. C. (2007). Labeling tetracysteine-tagged proteins with a SplAsH of color A modular approach to bis-arsenical fluorophores. Chembiochem 8, 1642-5. 

Gershon P.D., Khilko S., Stable chelating linkage for reversible immobilization of oligohistidine tagged proteins in the BIAcore surface plasmon resonance detector, J Immun Methods 1995 183 65-76. 

---