Fast purification of a functional elongator tRNAmet expressed from a synthetic gene in vivo.
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To study the interaction of an arninoacyl-tRNA synthetase with its cognate tRNAs, it is of interest to be able to design synthetic tRNA mutant genes, and to rapidly purify sufficient amounts of the corresponding tRNAs. A prerequisite is to show that the synthetase retains full specificity towards a wild type tRNA expressed from a synhetic gene. For this purpose, we have used an expression vector to overproduce and purify elongator tRNAmet. To date, the most widely used rapid purificaton procedure, extaction from polyacrylamide gels, does not provide enough pure material to achieve fine characterizations, such as affinity constants measurements by the analysis of the intrinsic enzyme fluorescence quenching (1). We propose a rapid method for the purification of a functional tRNAmet based on the construction of a synthetic gene, the obtention of an overproducing E. coli strain and a two-step FPLC driven purification from a crude extract. A shuttle vector, pBSTNAV, (derived from Bluescript M13+KS) carrying a multisite cloning sequence EcoRIISmaIlPstI flanked upstream with a synthetic lipoprotein promoter and downstream with the rrnC transcription terminator, both prepared from the pGFIB-I phasmid (2), was designed (figure The synthetic elongator tRNAmet gene constructed by assembling six different overlapping oligonucleotides synthetised with the Pharmacia gene assembler and purified on a Mono Q column. A mixture of the 6 oligonucleotides (1.5 pM each) was heated to 95 IC, slowly cooled and submitted to ligation during 1 hour at 37°C. Oligonucleotides 1 and 4 were kept unphosphorylated, to avoid the concatemerization of assembled