Dietrich Stueber

A T5 promoter-based transcription-translation system for the analysis of proteins in vitro and in vivo.

Publisher Summary This chapter discusses a T5 promoter-based transcription–translation system for analyzing proteins in vitro and in vivo . The chapter describes a simple method that permits the expression of cloned sequences in Escherichia coli as well as in cell-free in vitro systems of eukaryotic (wheat germ, reticulocyte, and HeLa cells) or prokaryotic ( E. coli ) origin, using a single expression unit. The expression of cloned genes in heterologous systems in vitro and in vivo has been instrumental in the identification and analysis of gene products and their derivatives. The essential element of the unit is a promoter derived from coliphage T5 that is utilized by E. coli ribonucleic acid (RNA) polymerase. It efficiently directs the synthesis of capped or uncapped mitochondrial RNA (mRNA) in vitro . Translation of such RNAs in the presence of [ 35 S] methionine yields single proteins of such high specific activity and purity that they can be directly analyzed by gel electrophoresis without the necessity of prior immunoprecipitation. The chapter also describes the application of the method for the study of structure/function relationships of proteins, including (1) protein synthesis in vivo and in vitro , (2) the translocation of proteins into and through membranes, and (3) the interruption of translation at predetermined sites for the generation and characterization of truncated proteins. Plasmid pDS5 and its derivatives 5 are members of a plasmid family developed for the study of transcriptional signals. The chapter also summarizes the essential properties of the pDS5 system.

Transcription from efficient promoters can interfere with plasmid replication and diminish expression of plasmid specified genes.

The copy number of plasmids containing the ColE1 replicon is affected by changes in the transcriptional activity within the plasmid if these changes lead to transcriptional readthrough into the replication region towards the promoter priming DNA replication. Such readthrough e.g., from the tet region in pBR322 not only causes overproduction of a peptide known to affect the copy number negatively but also appears to interfere negatively with the replication of the plasmid itself. The proper placement of efficient transcriptional terminators prevents such interference and permits the stable integration of strong promoters. Due to this termination effect, up to 9‐fold differences in plasmid copy number were observed, depending upon the particular growth conditions. The higher copy number is of course reflected by higher yields of plasmid‐specified gene products indicating the relevance of the above effects for studies of gene expression.

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

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.

Immunogenicity of recombinant Plasmodium falciparum rhoptry associated proteins 1 and 2

Mice and rabbits immunized with recombinant forms of malaria vaccine candidate antigens rhoptry‐associated proteins 1 and 2 (RAP‐1, RAP‐2 and rRAP‐l, rRAP‐2) produce antibodies at litres equivalent to monoclonal antibody ascites fluid raised against the native proteins. Sera from animals immunized with rRAP‐l contain antibodies which recognize the native protein by indirect immunofluorescence and immunoblotting, partially inhibit erythrocyte invasion in vitro and are long lasting. Epitope mapping shows these antibodies predominantly recognize epitopes in the N‐terminal third of rRAP‐l, some of which coincide with the targets of inhibitory monoclonal antibodies. By contrast, sera from animals immunized with rRAP‐2 contain antibodies which recognize the recombinant but not the native protein