Manfred Watzele

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.

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.

High-Throughput Expression PCR Used to Systematically Investigate Regulation of Translation Initiation in an E. coli Cell-Free Expression System

A number of publications describe strategies for optimization of heterologous protein expression in E. coli (an overview is given in Hannig and Makrides 1998). As the most important rate-limiting factor, the step of initiation of translation has been identified. Besides the start codon and a Shine-Dalgarno motive additional sequence characteristics seem to have an influence on the efficiency of translation initiation. Particularly important for translation initiation are sequence elements at the 5’-untranslated region of the mRNA. Makrides (1996) described different translational enhancer sequences such as, for example, a sequence from the T7-phage genel0 leader or a U-rich sequence from the 5 ’-untranslated region of some mRNAs like the atpE regulator from E. coli. The so-called downstream box, a sequence element of highly expressed T7 phage genes with homology to the ribosomal 16 S RNA, was also suggested as translational enhancer element (Sprengart et al. 1996). However, on the basis of this work, so far no universal expression vector could be proposed which is equally useful for every protein.