IN-VIVO DEUTERATION OF TRANSFER-RNAS - OVEREXPRESSION AND LARGE-SCALEPURIFICATION OF DEUTERATED SPECIFIC TRANSFER-RNAS

Structural investigations of tRNA complexes using NMR or neutron scatt ering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active for m. Fully deuterated tRNAs can be prepared from cells grown in deuterat ed minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of c ells. Here we describe the large-scale preparation of two deuterated t RNA species, namely (D)tRNA(Phe) and (D)tRNA(f)(Met) (the method is al so applicable for other tRNAs). Using overexpression constructs, the y ield of specific deuterated tRNAs is improved by a factor of two to te n, depending on the tRNA and growth condition tested. The tRNAs are pu rified using a combination of classical chromatography on an anion exc hange DEAE column with reversed phase preparative HPLC. Purification y ields nearly homogenous deuterated tRNAs with a chargeability of simil ar to 1400-1500 pmol amino acid/A(260) unit. The deuterated tRNAs are of excellent biological activity.