Traditionally, cell-free transcription/translation systems have been used mainly for analytical purposes to characterize gene products. Recently, this technology has become a powerful alternative to cell based methods to produce proteins. It exploits the availability of highly active cellular...

In this chapter we present a general protocol for producing small alpha helical membrane proteins in vitro suitable for X-ray crystallography. Increased protein solubility and highly efficient selenomethionine labeling makes this approach a convenient and relatively inexpensive method for...

Cell-free expression systems offer new possibilities to produce protein samples that are suitable for nuclear magnetic resonance (NMR) spectroscopy. Advantages of the cell-free approach include enhanced sample stability, minimal sample purification strategies and, especially, the prospect of...

Proteins provided with unique functional groups such as affinity labels or fluorescence moieties offer high potential in many biotechnological or biomedical investigations, e.g., immobilization studies or high throughput screenings. An attractive alternative to known posttranslational methods of...

In the broad area of functional proteomics, that is the global characterization of proteins and their function, cell-free rabbit reticulocyte lysate (RRL) has been used extensively to elucidate the mechanisms of mammalian translation, cotranslational modifications, post-translational...

A reconstituted protein synthesis system, comprising a minimal set of components for the production of active proteins, is described. The system consists of 32 purified soluble factors from Escherichia coli together with other biochemical components, and is designated as the PURE (Protein...

Protein microarray technology has empowered investigators with a tool that offers the potential to study the function of thousands of proteins in a single experiment. The approach typically involves a process by which proteins are tethered to either a modified microscope slide or to the bottom of...

The combination of rapid protein expression systems with high-throughput protein isolation methodologies leads to increased throughput in protein production, improves efficiency and promotes multiplexed high-throughput experimentation. Such pipelines gain wide importance in proteomic...

Cell-free protein synthesis systems have many advantages over conventional in vivo (cellular) expression. For example, they offer the potential for higher productivity, parallel production and simplified purification. Moreover, the openness of the cell-free system allows control of the reaction...

From the first procedure standardized in the early 1960s to its most recent modifications, cell-free expression using Escherichia coli lysates has evolved into a robust method for protein expression capable of producing protein in milligram quantities. Free from the constraints of a cell, the...

Global analysis of protein functions and networks has become the focus of considerable attention since the sequencing of the human genome. The development of proteomics has led to an increasing interest in cell-free translation systems because of their rapidity and ease of handling. We have...

A novel C-terminal labeling technology has been developed using fluorescently conjugated puromycin derivatives in cell free extracts. It is an easy to handle, rapid and low cost method for protein research. The labeling process requires only the addition of a labeling reagent to the translation...

Current, cell-free methodologies allow for robust and rapid protein production and screening. The use of protein arrays attached selectively or nonspecifically to various solid supports is rapidly becoming a common research tool to explore the function and potential relationships of proteins...

We have developed a toolbox approach for protein engineering to enhance protein expression yield and functionality by manipulating ribosomal “pause” signals correlated with over-represented codon pairs contained in the open reading frame of genes. CODA’s Translation Engineering algorithms...

Membrane proteins still represent one of the major challenges of structural biology. The preparation of high quality samples of functionally folded protein is usually the major bottleneck that restricts further approaches. Cell-free expression has emerged in recent times as a promising tool for...

Ribosome display is a powerful selection technology for the identification of proteins with desired functions and has wide applicability across biologics drug discovery and basic research. Ribosome display is based on polymerase chain reaction and cell-free translation technologies that combine...

This chapter is devoted to the emerging field of engineering artificial fluorescent proteins by using tRNA mediated expression labeling. The method of producing fluorescent proteins utilizes in vitro (cell-free) or in vivo (cellular) expression and relies on the use of engineered tRNAs...

Cell-free protein expression systems are becoming more widely used as they allow fast synthesis of recombinant proteins and an easy manipulation of reaction conditions. Here, we report on recent advances of a eukaryotic system based on extracts derived from Spodoptera frugiperda insect cells. We...