A novel in vitro transcription-translation system: accurate and efficient synthesis of single proteins from cloned DNA sequences.

Abstract

A system is described which permits the efficient synthesis of single proteins in vitro. The essential element in this expression system is a strong promoter derived from coliphage T5 which produces, with high efficiency, specific RNAs in capped or uncapped form, depending upon the experimental conditions used. The transcription‐coupled capping of RNA allows the direct translation of the RNA in eukaryotic extracts from wheat germ as well as from HeLa cells. The synthesis of three different proteins is reported, including lysozyme, which is shown to be translocated across membranes when appropriate assay conditions are used. The simplicity of the experimental procedure, the high purity and specific activity of the [35S]methionine‐labelled proteins produced offer a number of possibilities for the study of structure‐function relationships of proteins.

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

“…Radiolabeled precursor proteins were synthesized in reticulocyte lysate as described previously (Pelham and Jackson, 1976;Krieg and Melton, 1964;Stueber et al, 1964).…”

Characterization and in vitro expression of the cytochromeb-559 genes of barley. II. In vitro transcription and translation

“…Though, in these cases transcripts were "capped" by the addition of 7-methylguanosine triphosphate to the transcription mixture (18) no proteins were synthesized in the rabbit reticulocyte lysate and only low amounts of proteins were produced in the wheat germ extract (data not shown). $30 lysates prepared from intact chloroplasts (1) of spinach and barley did not show any protein synthesis due to exogenously added DNA or transcripts derived from pDS6 (data not shown).…”

“…DNA-fragments containing either both cytochrome b-559 genes together or the individual psbE and psbF were inserted into the multiple cloning sites of the expression vectors pDS6 (18) and pGem3 (Promega Biotec) (Figure 1). Transcripts made from pDS6 and pGem3 were translated in an $30 lysate prepared from E. coli cells (14,22).…”

“…In vitro transcription of DNA cloned into pDS6 or pDS6-RBSII was done according to STUEBER et al (18) but without capping of the RNA. 2 ~tg of supercoiled plasmid DNA were transcribed by 0.3 units of E. coli RNA-polymerase in a final volume of 10 ~tl containing 20 mM-HEPES/KOH, pH 7.5, 10 mM-Mg-acetate, 200 mM-K-acetate, 0.2 mM-spermidine, 5 mM-DTT, 10 units of RNasin (human placental RNase inhibitor, Boehringer) and 0.5 mM each of GTP, CTP, UTP and ATP.…”

“…In vitro expression of the recombinant pDS6 plasmid yielded only a polypeptide of about 4 kD molecular weight ( Figure 5) which is considered to be apoprotein II. Since STUEBER et al (18) had reported that the distance between the T5 promoter and the startcodon of inserted genes affects the efficiency of the system, the distance between the startcodon of the psbE gene and the T5 promoter was extended by 36 bp. This modification dramatically increased the synthesis of the 4 kD polypeptide but did not elicit the expression of the psbE gene ( Figure 5).…”