Puromycin

Puromycin is an antibiotic that is a protein synthesis inhibitor by inhibiting translation.

Inhibition of translation
Puromycin is an aminonucleoside antibiotic, derived from the Streptomyces alboniger bacterium, that causes premature chain termination during translation taking place in the ribosome. Part of the molecule resembles the 3' end of the aminoacylated tRNA. It enters the A site and transfers to the growing chain, causing premature chain release. The exact mechanism of action is unknown at this time, but, the 3' position contains an amide linkage instead of the normal ester linkage of tRNA, the amide bond makes the molecule much more resistant to hydrolysis and thus causes the ribosome to become stopped.

It is not selective for either prokaryotes or eukaryotes.

Also of note, puromycin is critical in mRNA display as it allows the growing peptide chain to be covalently bonded to its own mRNA template.

Peptidase Inhibitor
Puromycin is a reversible inhibitor of dipeptidyl-peptidase II (serine peptidase) and cytosol alanyl aminopeptidase. (metallopeptidase). The mechanism of inhibition is not well understood, however puromycin can be used to distinguish between aminopeptidase M (active) and cytosol alanyl aminopeptidase (inhibited by puromycin).

Cell culture
Puromycin is used in cell biology as selective agent in cell culture systems. It is toxic to prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the Pac gene encoding a puromycin N-acetyl-transferase (PAC) that was found in a Streptomyces producer strain. Puromycin is soluble in water (50 mg/ml) as colorless solution at 10 mg/ml. Puromycin is stable for one year as solution when stored at -20°C. The recommended dose as a selection agent in cell cultures is within a range of 1-10 μg/ml, although it can be toxic to eukaryotic cells at concentrations as low as 1 μg/ml. It acts quickly and can kill up to 99% of nonresistant cells within 2 days.

Selection of Escherichia coli
Puromycin is poorly active on E. coli. Puromycin-resistant transformants are selected in LB agar medium supplemented with 125 µg/ml of puromycin. But use of puromycin for E. coli selection requires precise pH adjustment and also depends on which strain is selected. For hassle–free selection and optimum results the use of special modified puromycin is possible. Plates containing puromycin are stable for 1 month when stored at 4°C.