Cordula Nemetz

Rapid translation system: A novel cell-free way from gene to protein

Proteome research has recently been stimulated by important technological advances in the field of recombinant protein expression. One major breakthrough was the development of a new generation of cell-free transcription/translation systems. The open and flexible character of these systems allows direct control over expression conditions via the addition of supplements to the expression reaction. The possibility of working with linear expression templates instead of cloned plasmids and the ease of downstream processing, circumventing the need for cell-lysis, makes them ideally suited for high-throughput screening applications. Among these novel cell-free systems, the Rapid Translation System (RTS) developed by Roche is the first one that is scalable from micrograms to milligrams of protein. This review describes the basic principles of RTS which differentiate it from traditional in vitro expression technologies, starting from template generation to high-end applications like labeling for structural biology research. Recent results obtained by RTS users from different institutions are presented to illustrate each step of a novel cell-free protein expression workflow and its benefits compared to traditional cell-based expression.

Reliable quantification of in vitro synthesized green fluorescent protein: Comparison of fluorescence activity and total protein levels

At any time in vitroor in vivoexpressed unlabeled proteins have to be quantified it is difficult to find a reliable method, especially with nonpurified samples. Quantification viaprotein activity can result in too low levels if the proteins analyzed tend to aggregate into inactive forms. Here, wild‐type green fluorescent protein (GFPwt) was expressed in high amounts in vitrousing the Rapid Translation System 500 based on Escherichia colilysates. Fluorescent activity was determined in dependence of oxygen and compared to total protein levels. In the presence of low amounts of oxygen only 16% of the whole GFPwt amounts were detectable viadetermination of fluorescence activity. A reliable method to easily quantify whole protein levels even without specific antibodies and without purification steps by simple sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and Coomassie blue staining is described.

Optimization of the Translation Initiation Region of Prokaryotic Expression Vectors: High Yield In Vitro Protein Expression and mRNA Folding

The process of in vitro protein synthesis that is based on T7 RNA polymerases differs strongly from what occurs in E. coli (Studier et al. 1990). Since the RNA polymerase of bacteriophage T7 works more than five times faster than endogenous RNA polymerases and the E. coli translation machinery, in vitro synthesized mRNAs are less protected by bacterial ribosomes. Consequently, no real coupling of prokaryotic transcription and translation can take place in vitro (Spirin 1999). As unprotected mRNAs easily form secondary structures, double-stranded regions can block the accessibility of important regulatory elements like the ribosomal binding site (RBS or Shine-Dalgarno site) and the start codon (AUG) and, thereby, inhibit the initiation of translation. Here, the initial region of prokaryotic expression vectors containing a T7 promoter and a T7 gene 10 enhancer was investigated and optimized for in vitro protein expression reactions.

Generation of Linear Expression Elements by PCR

Standard methods for coupled in vitro transcription and translation were formerly limited by the need for cloning the gene of interest into an appropriate expression vector, subsequent bacterial amplification, DNA extraction and purification and final restriction enzyme digestion of the desired DNA template. Nowadays, it is a well-established process to amplify the coding region of a gene or a cDNA by PCR and use the linear product as a template for direct in vitro expression of the encoded protein.

Cell-Free Expression of Soluble Human Erythropoietin

Erythropoietin (EPO) is a haematopoietic hormone produced by the kidney and secreted into the bloodstream to stimulate self-renewal and differentiation of late erythroid precursor cells toward mature red blood cells (Graber and Krantz 1978). As with many circulating hormones, EPO is highly glycosylated at conserved sites. It contains four complex carbohydrate chains, which have been implicated in the biological activity and stabilization of the protein (Goldwasser et al. 1974, Takeuchi and Kobata 1991). A possible role of these sugar chains for the correct folding and the solubility of EPO has been proposed, but this glycosylation is not necessary for binding of EPO to the specific EPO receptor (Delorme et al. 1992).

High-Level Cell-Free Protein Expression from PCR-Generated DNA Templates

Cell-free DNA-dependent in vitro transcription/translation is a well-established procedure when working with the expression of circular closed DNA and with long linear DNA. Attempts of expression from short pieces of linear DNA were only partially successful. The smaller the DNA used, the more difficult it was to produce relevant amounts of protein. It was shown that these difficulties mainly resulted from the presence of exonucleases. During in vitro transcription and translation with S30 lysates from Escherichia coli, it was demonstrated that exonuclease V was responsible for the degradation of linear DNA. Exonuclease V consists of three subunits (the gene products of recB, recC, recD). This exonuclease cleaves linear DNA from its 3′-ends.

Rapid Protein Engineering by Expression-PCR

With the start of the postgenomic era, it is an important issue to analyze the proteins encoded by the enormous sequence data collected from genome projects. For studies, e.g., concerning the structure-function relationships of proteins, the corresponding genes are often mutated, using procedures based on the polymerase chain reaction. This proved to be a highly versatile process adaptable to a wide range of procedures and applications. PCR mutagenesis procedures make it possible to modify and engineer any target DNA with ease and high efficiency. This includes, for example, the introduction of point mutations, deletions or insertions, and approaches for domain fusion and random mutagenesis (Newton and Graham 1997; McPherson and Moller 2000). The basic procedure that is adapted for the introduction of point mutations is described as two-sided splicing by overlap extension (Horton et al. 1989). The method is very efficient and specific. As linear expression constructs can directly be expressed in vitro it is indicated to combine expression-PCR (E-PCR) with the PCR mutagenesis methods already known.