Bernd Buchberger

Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ ions.

AbstractPurpose. One of the drawbacks of polycationic and cationic liposomalgene transfer is its sensitivity to serum. Gene therapy requires thetransfectant-DNA complex to be resistant to serum as well as blood.Since Ca2+ has proved to be an efficient cofactor of polycationic genetransfer, we decided to investigate its effects on transfection in thepresence of serum. Methods. We studied transgene expression of luciferase gene (pCMVLuc) on ECV 304 human endothelial cells using H1 histone andDOSPER as transfectants in the presence of 0-100% fetal calf serum. Results. H1-and DOSPER-mediated transfection was found to beinhibited by serum above the concentration of 10%. If 2 mM Ca2+ or2 mM Ca2+/0.1 mM chloroquine was included in the culture mediumwhich replace the transfection mixture and was left on the cells for24 hours postincubation, the inhibiting effect of even 100% serumwas overcome. Conclusions. A high serum level does not interfere with binding anduptake of H1- and DOSPER-DNA complexes, but inhibits subsequentsteps such as endosomal escape. Ca2+ in the form of nascent calciumphosphate microprecipitates and other lysosomolytical agents facilitateendosomal/lysosomal release by their fusigenic and membranolyticactivity.

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.

A NEW EFFICIENT METHOD FOR TRANSFECTION OF NEONATAL CARDIOMYOCYTES USING HISTONE H1 IN COMBINATION WITH DOSPER LIPOSOMAL TRANSFECTION REAGENT

Although cationic lipids are successfully used for gene transfer in vitro, primary cells such as neonatal cardiomyocytes frequently resist efficient transfection. We show here that the polycationic lipid DOSPER in combination with histone H1 was much more efficient in transfection of neonatal cardiomyocytes than DOSPER alone or other cationic lipids. This has been shown for transfection with the reporter plasmids pSV β-gal and pCMV luc. If viral transfections are not possible, this mild method is an alternative to transfect cardiomyocytes.

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.

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.

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.

Matrix Reactor: A New Scalable Reactor Principle for Cell-Free Protein Expression

For a long time, cell-free protein synthesis was more or less an analytical method due to low synthesis rate and productivity. However, several key improvements over the last decades have lead to protocols providing real preparative amounts of protein: 1. Coupling of transcription/translation using DNA templates and exogenous phage RNA-polymerases (Zubay 1973, Spirin 1992, Craig et al. 1992) 2. Reactor technologies using continuous supply of substrates/continuous removal of products: Continuous exchange cell-free (CECF) or continuous flow cell-free (CFCF) systems by Spirin et al. (1988) 3. Optimization of biochemistry of lysate systems (Baranov and Spirin 1993, Kim et al. 1996, Kim and Swartz 1999, Madin et al. 2000).