Vectors for cell-free expression and mutagenesis of protein-coding sequences.

We have developed a vector (pTL-8) to facilitate cell-free expression of cloned protein-coding sequences (1). The 5' non-coding and initial coding sequences of the tobacco etch virus (TEV) RNA genome were inserted downstream from an SP6 promoter, allowing foreign coding sequences to utilize the translational initiation properties of the TEV leader for cell-free translation from synthetic transcripts. Efficient and accurate initiation of translation occurs without the need for 5' capping procedures (1). A family of vectors has been developed which increases the applicability of pTL-8 and which allows single-stranded DNA production for oligonucleotide-directed mutagenesis. Vector pTL-8 contains the cDNA for the TEV leader segment inserted into pGEM-4 (Promega-Biotech), while pTL-17 harbors the leader segment in pGEM-3; thus, transcription [using standard protocols, (2)] from the SP6 promoter in pTL-8 derivatives or from the T7 promoter in pTL-17 derivatives results in (+)-strand synthesis. Relative to the TEV leader sequence, the orientation and translational reading frame of the polylinker in both vectors are the same. Vectors pTL-8S and pTL-37 (shown below) contain the M13 origin of replication (Aal W-Nar I fragment from pUC118) inserted between the Aat II and Nar I sites of pTL-8 and pTL-17, respectively. Cloning of these plasmids in E. coli F strains and transfection with the helper phage M13K07 allows single-stranded DNA production [(+)-sense for pTL-8S, (-)-sense for pTL-37.]