Puromycin conjugates for specific C-terminal protein labeling in vitro and monitoring of global protein synthesis in vivo

The antibiotic puromycin is a structural analog of aminoacyl-tRNA that has been traditionally used as an inhibitor of protein synthesis since it specifically incorporates at the C-terminus of nascent polypeptide chains thereby stopping translation. Based on this observation, a new and significantly faster approach, using labeled puromycin conjugates, can be applied to both, specific C-terminal labeling of full-length protein in vitro^[1,2,3,4] and non-radioactive monitoring of global protein synthesis in vivo^[5,6,7].

Labeled dC-puromycin conjugates: Specific C-terminal protein labeling of full-length protein in vitro
C-terminal labeled full-length protein can be produced by in vitro translation in the presence of low concentrations of labeled dC-puromycin conjugates^[1].
These proteins have been successfully used to analyze protein-protein interactions (pull-down assay, FCCS, protein-protein microarrays) and protein-DNA interactions (DNA microarrays)^[2,3,4].
In contrast to traditionally applied posttranslational labeling approaches, additional time consuming purification steps can be avoided due to the synchronization of protein expression and labeling.

O-Propargyl-puromycin: Monitoring of global protein synthesis in vivo
Current studies of the cellular protein level rely on indirect methods such as DNA and mRNA microarrays or classical radioactive metabolic labeling with ³⁵S-methionine.
O-Propargyl-puromycin is cell-permeable and readily incorporated into newly translated proteins thus providing a sensitive, nonradioactive method for direct monitoring of global protein synthesis^[5].
The resulting C-terminal alkyne labeled proteins can be detected via Cu(I)-catalyzed click chemistry that offers the choice to introduce a Biotin group (Azides of Biotin → Click Chemistry) for subsequent purification tasks or a fluorescent group (Azides of fluorescent dyes → Click Chemistry) for subsequent microscopic imaging.

Selected References

[1] Miyamoto-Sato et al. (2000) Specific bonding of puromycin to full-length protein at the C-terminus. Nucleic Acids Res. 28 (5):1176.
[2] Nemoto et al. (1999) Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems. FEBS Letters 462:43.
[3] Doi et al. (2002) Novel Fluorescence Labeling and High-Throughput Assay Technologies for In Vitro Analysis of Protein Interactions. Genome Research 12:487.
[4] Kawahashi et al. (2007) High-throughput fluorescence labeling of full-length cDNA products based on a reconstituted translation system. J. Biochem. 141 (1):19.
[5] Liu et al. (2012) Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin. Proc Natl Acad Sci USA. 109 (2):413.
[6] Stark et al. (2004) A General Approach to Detect Protein Expression In Vivo Using Fluorescent Puromycin Conjugates. Chemistry & Biology 11:999.
[7] Godman et al. (2011) Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. The FASEB Journal 25:1028.