Selective Dye-Labeling of Newly Synthesized Proteins in Bacterial Cells

Abstract

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,

Fluorogenic click reaction

“…4b). 28,29 They also exploited their work to label newly synthesized proteins in vivo by tagging mammalian cells with Hpg for several hours, and then treated the cells with membrane permeable 3-azido-7-hydroxycoumarin 13 overnight in the dark in the presence of CuSO 4 , tris(carboxyethyl)-phosphine (TCEP) and TBTA. Mammalian cells of different kinds (fibroblasts, epithelial and endothelial cells) and from different species (mouse, monkey, hamster and human) have been studied and showed very strong fluorescence in some parts of the cell structure.…”

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

“…The coumarin dye as a profluorophore can produce a strong fluorescence when a triazole ring is formed by azide-alkyne cycloaddition (Fig. 3a) (35)(36)(37). When the coumarin dye-labeled probe is free in the solution (or in the cell) without G-quadruplex formation, two molecules (azidocoumarin and alkyne at the 5Ј end) are separated spatially, and no fluorescence is observed.…”

Polysaccharides: The “Click” Chemistry Impact

“…The CuAAC was applied to biological systems such as virus particles [65] and Escherichia coli cells [66][67][68][69] quite early on. Considering that the harmless introduction of modified sugars in glycans had already been studied, [70,71] the "click" chemistry was naturally employed for its mild conditions and its short reaction time.…”