Resto E, Iida A, Van Cleve M D, Hecht S M
Department of Chemistry, University of Virginia, Charlottesville 22901.
Nucleic Acids Res. 1992 Nov 25;20(22):5979-83. doi: 10.1093/nar/20.22.5979.
Large quantities of a catalytically active protein have been produced in a cell free system. More than 10(9) copies of protein were produced from each DNA plasmid containing DNAfol, the bacterial gene encoding dihydrofolate reductase (DHFR). The strategy employed, denoted gene amplification with transcription/translation (GATT), involves sequential coupling of (i) DNA amplification by the polymerase chain reaction (PCR) and (ii) in vitro RNA transcription by T7 RNA polymerase, followed by (iii) translation of the run-off transcripts in a rabbit reticulocyte system. The protein product had the expected size (18 kDa) and catalyzed the NADPH-dependent reduction of 7,8-dihydrofolic acid to 5,6,7,8-tetrahydrofolic acid as efficiently as authentic DHFR. Potential applications of the strategy include large scale production of enzymes containing synthetic amino acids and facilitation of the characterization of the function of genes encountered in genomic mapping studies.
在无细胞系统中已产生了大量具有催化活性的蛋白质。从每个含有DNAfol（编码二氢叶酸还原酶（DHFR）的细菌基因）的DNA质粒中产生了超过10⁹个蛋白质拷贝。所采用的策略称为转录/翻译基因扩增（GATT），它涉及以下步骤的顺序耦合：（i）通过聚合酶链反应（PCR）进行DNA扩增，（ii）通过T7 RNA聚合酶进行体外RNA转录，然后（iii）在兔网织红细胞系统中对延伸转录本进行翻译。蛋白质产物具有预期大小（18 kDa），并且能像天然DHFR一样高效地催化NADPH依赖的7,8 - 二氢叶酸还原为5,6,7,8 - 四氢叶酸。该策略的潜在应用包括大规模生产含合成氨基酸的酶以及促进基因组图谱研究中所遇到基因功能的表征。
