Jünemann R, Wadzack J, Triana-Alonso F J, Bittner J U, Caillet J, Meinnel T, Vanatalu K, Nierhaus K H
Max-Planck-Institut für Molekulare Genetik, Berlin-Dahlem, Germany.
Nucleic Acids Res. 1996 Mar 1;24(5):907-13. doi: 10.1093/nar/24.5.907.
Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely D-tRNAPhe and D-tRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity.
使用核磁共振（NMR）或中子散射对tRNA复合物进行结构研究通常需要氘代的特定tRNA。这些tRNA需要大量的、高度纯化且具有生物活性的形式。完全氘代的tRNA可以从在氘代基本培养基中生长的细胞制备，但在这种条件下tRNA含量较低，这是由于tRNA生物合成会响应细胞的缓慢生长而受到调控。在这里，我们描述了两种氘代tRNA物种，即D - tRNAPhe和D - tRNAfMet的大规模制备方法（该方法也适用于其他tRNA）。使用过表达构建体，特定氘代tRNA的产量提高了2至10倍，这取决于所测试的tRNA和生长条件。通过在阴离子交换DEAE柱上进行经典色谱法与反相制备型高效液相色谱法相结合的方式来纯化tRNA。纯化后可得到几乎均一的氘代tRNA，其每A260单位的氨基酸充电能力为1400 - 1500皮摩尔。氘代tRNA具有优异的生物活性。
