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

Abstract

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.

Molecular genetic analyses of the soybean mosaic virus NIa proteinase

“…This is the predicted size of precursor which has not been proteolytically processed. Similar mutation and expression studies have been reported for the TEV N I a proteinase (Carrington & Dougherty, 1987) and are presented in Fig. 4 (b).…”

“…A portion of pSMV-34 was subcloned into the transcription vector pTL-37 (Carrington et al, 1987). This plasmid, referred to as pTL-TSP, was generated by digesting pSMV-34 with NeoI, filling in using Klenow fragment, digesting with XbaI and ligating into pTL-37 which had been digested with Sinai and Xbal.…”

“…In order to determine whether a proteolytic activity was encoded by this region, nearly all of the SMV NIa proteinase coding sequence plus additional downstream sequences that encoded the NIa/NIb cleavage junction and the amino terminal 9K of the NIb protein were inserted in the transcription vector pTL-37 (Carrington et al, 1987). The resulting recombinant (Robaglia et al, 1989), PPV (Maiss et al, 1989), TEV (Allison et al, 1986) and TVMV (Domier et al, 1986).…”

Cleavage profiles of tobacco etch virus (TEV)-derived substrates mediated by precursor and processed forms of the TEV NIa proteinase

“…Standard recombinant DNA techniques (Sambrook et al, 1989) were used to insert the PCR-generated products described above into the transcription vector pTL27N. This plasmid vector was similar to pTL17 described previously (Carrington et al, 1987), except that an origin of replication for single-stranded DNA production was inserted and a site-directed mutation creating an NcoI restriction site at the ATG translation start codon (TEV nucleotides 143 to 145) was introduced (Taylor et al, 1985a, b).…”

Autocatalytic activity of the tobacco etch virus NIa proteinase in viral and foreign protein sequences

“…1 (b) and are referred to as pTL-50/71, pTL-58/30 and pTL-combo. All three plasmids positioned the coding sequences downstream of a TEV 5' leader cassette to promote efficient translation in rabbit reticulocyte lysates (Carrington et al, 1987).…”