Playing tag: Noncanonical amino acid tagging enables the selective fluorescent visualization of newly synthesized proteins in mammalian cells (see the picture). Susceptibility to tagging is determined by the spatial and temporal character of the protein synthesis, thus providing a complement to methods which identify relevant members of the proteome.

Plasmid and Expression Hosts. The plasmid pQE30-Barstar contains a gene encoding histidine-tagged barstar under control of a T5 promoter. Briefly, PCR was used to add BamHI and Hind III sites to the barstar gene. The gene encodes two mutations (Cys53Ala, Cys95Ala) for improved stability that do not affect barnase binding 1 . We do not believe that cysteine deletion is necessary for effective labeling; we have observed labeling of many cysteine-bearing proteins in companion experiments. After digestion, the gene was inserted into pQE30 between the BamHI and HindIII restriction sites. For incorporation of homopropargylglycine (Hpg), pQE30-Barstar was transformed into the E. coli methionine auxotrophic strain M15-MA to make the expression host M15-MA [pQE30-Barstar]. 2 For incorporation of ethynylphenylalanine (Eth), pQE30-Barstar was linearized by digestion with NheI. The plasmid pQE15-PheRS* contains a mutant E. coli phenylalanyl-tRNA synthetase (A294G) under control of a modified tac promoter with an abolished lac repressor binding site for constitutive expression. 3, 4 This plasmid was digested with NheI, and a 1 kB fragment corresponding to the PheRS* cassette was isolated by agarose gel electrophoresis. This fragment was ligated into pQE30-Barstar to yield the plasmid pQE30-Barstar-PheRS*. This plasmid was transformed into the phenylalanine auxotrophic E. coli strain BL21 (DE3) After reaching an OD 600 of 1, the culture was sedimented by centrifugation for 5 minutes (6500g) at 4 ˚C. The cell pellets were washed twice with NaCl (0.9 wt %). The culture was resuspended in M9 minimal medium without methionine. The culture was divided into 35, 48, and 5 mL aliquots. For protein synthesis inhibition, tetracycline (10 mg/mL) was added to the 5 mL culture. After 15 minutes at 37 ˚C, the samples were supplemented with either methionine (0.75 mM in 35 mL medium) or Hpg (1 mM in 48 mL medium). After 10 minute incubation, protein expression was induced for 3.5 h by the addition of IPTG (1 mM). The 5 mL culture supplemented with Hpg and tetracycline was not induced.The expression of barstar from AF-IQ [pQE30-Barstar-PheRS*] was performed as above, except the M9 minimal medium was supplemented with chloramphenicol (35 mg/L) instead of kanamycin. After reaching an OD 600 of 1.0, a medium shift was performed as described. The cultures were supplemented with either phenylalanine (1.5 mM) or Eth (1.5 mM). Expression was induced for 3.5 h with IPTG (1 mM).(1) Ramachandran, S.; Udgaonkar,

Metabolic labeling of proteins with the methionine (1) surrogate azidonorleucine (2) can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected.

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