Puromycin dihydrochloride is produced by Streptomyces alboniger, a grampositive actinomycete, through a series of enzymatic reactions.[1] Puromycin dihydrochloride included a nucleoside covalently bound to an amino acid, mimicking the 30 end of aminoacylated tRNAs that participate in delivery of amino acids to elongating ribosomes.[2] It inhibits the growth of animal cells and blocks protein synthesis by binding to 80S ribosomes at low doses.[3]
In vitro study determined the optimal concentration of Puromycin dihydrochloride for selecting EGFPac-transfected cells by performing a Puromycin dihydrochloride resistance test. The puromycin-resistant gene (termed pac) encoding puromycin N-acetyl transferase was isolated from Streptomyces aboniger. If pac is introduced and expressed in animal cells, the cells can survive in the presence of Puromycin dihydrochloride. Results ahowed that it could successfully produce a somatically cloned transgenic piglet using recombinant cells obtained after gene transfer of a transgene (carrying both EGFP and pac expression units) and subsequent in vitro selection with a low concentration (2 mg/ml) of puromycin.[3]
In vivo study was conducted to determine the surface sensing of translation (SUnSET) technique could be used to measure the protein synthesis in whole tissues. Since there is currently an intense interest in identifying the molecular mechanisms that regulate skeletal muscle protein synthesis. It allows for the visualization and quantification of protein synthesis and eliminates the need for generating radioactive tissues/animals. This study also determined that the surface sensing of translation could detect relatively acute changes in protein synthesis in the absence of changes in rRNA as well as detect not only increases but also decreases in protein synthesis in vivo. [4]
References:
[1]. Tercero JA, Espinosa JC, Lacalle RA, Jiménez A. The biosynthetic pathway of the aminonucleoside antibiotic puromycin, as deduced from the molecular analysis of the pur cluster of Streptomyces alboniger. J Biol Chem 1996;271(3):1579–90.
[2]. Aviner R. et al. The science of puromycin: From studies of ribosome function to applications in biotechnology. Comput Struct Biotechnol J. 2020 Apr 24;18:1074-1083.
[3]. Watanabe S, Iwamoto M, et al. A novel method for the production of transgenic cloned pigs: electroporation-mediated gene transfer to non-cultured cells and subsequent selection with puromycin. Biol Reprod. 2005 Feb;72(2):309-15.
[4]. Goodman CA, Mabrey DM, et al. Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new nonradioactive in vivo technique. FASEB J. 2011 Mar;25(3):1028-39.
Puromycin二盐酸盐是由革兰氏阳性放线菌Streptomyces alboniger通过一系列酶反应产生的。它包含一个核苷酸共价结合到一个氨基酸上,模拟参与将氨基酸传递给伸长中的核糖体的30端氨基acylated tRNAs。在低剂量下,它抑制动物细胞的生长并通过结合80S核糖体阻止蛋白质合成。
通过进行Puromycin dihydrochloride抗性测试,体外研究确定了选择EGFPac转染细胞的最佳浓度。从链霉菌中分离出编码puromycin N-乙酰转移酶的耐普罗霉素基因（称为pac）。如果将pac引入和表达在动物细胞中,则这些细胞可以在存在Puromycin dihydrochloride的情况下存活。结果显示,在使用低浓度（2 mg/ml）普罗霉素进行体外筛选后,成功地产生了一个体细胞克隆转基因小猪,该小猪是通过基因转移EGFP和pac表达单元的重组细胞获得并进一步筛选而来的。[3]
这篇文章介绍了一项在体内进行的研究,旨在确定表面感知翻译（SUnSET）技术是否可用于测量整个组织中的蛋白质合成。由于目前人们对骨骼肌蛋白质合成调节分子机制的兴趣非常高,该技术可以实现蛋白质合成的可视化和定量,并消除了产生放射性组织/动物的需要。此外,该研究还确定了表面感知翻译可以检测到相对急性的蛋白质合成变化,在rRNA没有改变时也能检测到,并且不仅能够检测到增加而且还能够检测到减少体内蛋白质合成。