Extracted from Streptomyces alboniger bacteria, Puromycin is an aminonucleoside antibiotic that inhibits protein synthesis by interfering with the translation process. It specifically inhibits peptidyl transfer on both prokaryotic and eukaryotic ribosomes. The antibiotic inhibits the growth of Gram positive bacteria and various animal and insect cells. Fungi and Gram negative bacteria are resistant to Puromycin due to its low permeability. But in some particular conditions puromycin can be used for E. coli. For more than 30 years, puromycin has been widely used as a basic tool for studying protein synthesis. Now, puromycin hydrochloride is particularly useful for the selection of cell types harbouring plasmids carrying puromycin resistance genes. ;RESISTANCE TO PUROMYCIN;

The pac gene encoding a Puromycin N-acetyl-transferase (PAC) has been isolated from a Streptomyces productin strain^1,2. It is located in a region of the pur cluster linked to the other genes determining the puromycin biosynthetic pathway. The expression of pac gene confers puromycin resistance to transfected mammalian cells³ expressing it. Under some conditions puromycin could also be used for selection of E. coli strains transformed with plasmids carrying the pac gene.

CONDITIONS OF SELECTION

Puromycin is poorly active on E. coli but is particularly useful in experiments conducted with mammalian cells. It can be used as an alternative to the Neomycin system for transfection experiments.

Mammalian cells

The working concentrations of puromycin for mammalian cell lines range from 1 to 10µg/mL. In a starting experiment we recommend to determine optimal concentrations of antibiotic required to kill your host cell line. Puromycin quickly kills eukaryotic cells that do not contain the pac gene. Dying cells detach form the plates allowing easy and early identification of transformant clones.

References:

1) VARA J., et al. (1985). Biochemistry. 24: 8074-8081

2) LACALLE R.A., et al. (1989). Gene. 79: 375-380

3) DE LA LUNA S and J. ORTIN. (1992). Methods In Enzymology. 216: 376-385

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