Wolfgang Stiege

The potentials of the in vitro protein biosynthesis system

Recent developments indicate that with the in vitro protein biosynthesis system a new technology emerges, which will find in the future its application in biotechnology. Up to date the best system described produces 2 mg of proteins per 0.5 ml after 100 h. The potentials of the in vitro protein biosynthesis system include not only the production of proteins, but especially the synthesis of proteins, which are toxic to living cells, or proteins with unnaturally modified or isotope-labelled amino acids in specific positions. Among the advantages of the in vitro system, when compared to current cloning techniques, are the purity of the proteins synthesized and their superior biological activities. Thus, the application of this technology will be manifold ranging from the production of proteins with improved or even new characteristics to the potentials of improving the methods of protein design and structural characterization.

In Vitro Translation in an Insect-Based Cell-Free System

The increasing number of protein sequences without attributed function which are continuously being discovered in various genome-sequencing projects demands the development of effective protein synthesis systems. Cell-free protein expression methods provide powerful tools to synthesize any desired protein, including native proteins, proteins toxic to living cells and artificially modified proteins. Eukaryotic in vitro translation systems in particular, have generated increased interest in their use in tackling fundamental problems in biochemistry and pharmacology. Rabbit reticulocyte lysate and wheat germ extract represent such systems, and these are widely used to characterize proteins and investigate mRNA translational mechanisms (Pelham and Jackson 1976; Erickson and Blobel 1983; Jackson and Hunt 1983; Madin et al. 2000). Additional lysates prepared from mammalian sources, such as Ehrlich ascite cells, human HeLa or mouse L-cells, have also given us efficient tools to study the synthesis and cell-free assembly of multiple proteins, in particular the generation of virus particles from mRNA in vitro (Molla et al. 1991; Bergamini et al. 2000). Thus, the rapid development of cell-free translation systems from a variety of cell lines is reaching the next stage, i.e., the large-scale production of eukaryotic lysates displaying properties which are optimized for individual purposes. In this chapter we describe such a system and detail how we have taken a widely used in vivo expression system, based on baculovirus-infected insect cells, and developed a standardized method for production of lysates which are suitable for in vitro translation.

Cell-free synthesis of functional and endotoxin-free antibody Fab fragments by translocation into microsomes

A eukaryotic cell-free system based on Spodoptera frugiperda cells was developed for the convenient synthesis of Fab antibody fragments and other disulfide bridge containing proteins. The system uses (i) a cell lysate that is mildly prepared under slightly reduced conditions, thus maintaining the activity of vesicles derived from the endoplasmic reticulum, (ii) signal peptide dependent translocation into these vesicles, and (iii) a redox potential based on reduced and oxidized glutathione. Monomeric heavy and light immunoglobulin chains are almost completely converted to highly active dimeric Fab joined by intermolecular disulfide bridges without supplementation of chaperones or protein disulfide isomerase. The applicability of the system is demonstrated by the synthesis of anti-lysozyme and anti-CD4 Fab antibody fragments yielding approximately 10 µg Fab per milliliter reaction mixture. The lack of endotoxins in this system is a prerequisite that synthesized Fab can be applied directly using whole synthesis reactions in cell-based assays that are sensitive to this substance class. Moreover, the system is compatible with PCR-generated linear templates enabling automated generation of antibody fragments in a high-throughput manner, and facilitating its application for screening and validation purposes.

Chapter 2 In Vitro Synthesis of Posttranslationally Modified Membrane Proteins

Publisher Summary Membrane proteins have become an important focus of the current efforts in structural and functional genomics and the rapid progress of various genome sequencing projects has greatly accelerated the discovery of novel genes encoding membrane proteins. In contrast, the molecular analysis of membrane proteins lags far behind that of cytosolic soluble proteins. Preparing high quality samples of functionally folded proteins represents a major bottleneck that restricts further structural and functional studies. Cell-free protein expression systems, in particular those of eukaryotic origin, have recently been developed as promising tools for the rapid and efficient production of a wide variety of membrane proteins. A large number of these proteins, however, require posttranslational modifications for optimum function. Several membrane proteins have been expressed in vivo to date, most of them being functionally, antigenically, and immunogenically similar to their authentic counterparts. This is mainly because of the properties of cultured eukaryotic cells, which are able to carry out many types of posttranslational modifications, such as the addition of N- and O-linked oligosaccharides, but also palmitoylation, myristylation, and phosphorylation.

A novel in vitro translation system based on insect cells

Background Various genome sequencing projects are identifying many new protein sequences but it is key to attribute functions to these proteins. A huge number of proteins have been expressed in vivo to date, most of them being functionally, antigenically and immunogenically similar to their authentic counterparts. This is mainly due to the properties of cultured eukaryotic cells, which are able to carry out many types of posttranslational modifications such as addition of Nand O-linked oligosaccharides, but also palmitoylation, myristylation and phosphorylation.

An Improved Protein Bioreactor Efficient Product Isolation During in vitro Protein Biosynthesis Via Affinity Tag

In vitro protein biosynthesis became a powerful technology for biochemical research. Beside the determination of structure and function in vitro selection of proteins is also of great interest. In most cases the use of a synthesized protein for further applications depends on its purity. For this purpose the in vitro production and purification of proteins with short affinity tails was established. A cell-free protein synthesis system was employed to produce bovine heart fatty acid-binding protein and bacterial chloramphenicol acetyltransferase with and without fusion of the Strep-tag affinity peptide. The quantitative removal of fusion protein during cell-free synthesis from a batch reaction and a semicontinuous flow cell-free reactor were achieved. No significant influence of the Strep-tag and the conditions during the affinity chromatography on maturation or activity of the proteins were observed. The product removal from the continuous flow cell-free reactor is still an only partially solved problem, because the use of ultrafiltration membranes has some limitations. The results document that it should be possible to avoid these limitations by introducing an affinity system.

RIBONUCLEOLYTIC ACTIVITIES IN THE ESCHERICHIA COLI IN VITRO TRANSLATION SYSTEM AND IN ITS SEPARATE COMPONENTS

mRNA stability is a limiting parameter for the efficiency of in vitro protein biosynthesis. In order to develop strategies to prolong the mRNA half‐life, we investigated the ribonuclease activities in the complete Escherichia coli system, in the separate cell fractions 70S ribosomes and S‐100 and in the non‐cellular fraction. Our results imply that the amount of ribonucleolytic activities and the insensitivity to placental RNase inhibitor in the complete system are due to the 70S ribosome fraction, whereas the generation of small degradation products is due to the S‐100 fraction. Remarkably, the human placental RNase inhibitor is able to reduce mRNA degradation in the bacterial S‐100 fraction.

Recombinant protein production in cell-free systems: strategies for improving yield and functionality

Background Despite the widespread use of CHO cells for the production of many industrially relevant biopharmaceuticals, this system is poorly understood on the genetic level and mainly relies on empirical procedures, due to the lack of adequate sequence information. In order to advance its overall performance, we successfully tested the applicability of a cross-species microarray approach, for investigating CHO specific transcription profiles [1]. In the present study we show expression signatures of individual recombinant CHO clones which correlate with their associated phenotype.

Towards Improved Applications of Cell-Free Protein Biosynthesis - The Influence of mRNA Structure and Suppressor tRNAS on the Efficiency of the System

The cell-free protein biosynthesis has the potential to become a powerful technology for the biochemical research in particular in the determination of the structure and function of proteins. The number of possible applications is rising with the obtainable yields and with the expanded feasibility of introducing modified amino acids into proteins. Here we describe the influence of two RNA translation components, the mRNA and the suppressor tRNA, on the efficiency of protein biosynthesis.