Chapter category: Cell Metabolism
Translation Engineering and Synthetic Biology
Cell-Free Protein Expression
Edited by: Wieslaw Kudlicki, Federico Katzen and Robert BennettISBN: 978-1-58706-123-3
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Chapter authors:
David A. Roth, Liza S.Z. Larsen and G. Wesley Hatfield
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David A. Roth
Research and Development, CODA Genomics, Inc.
Liza S.Z. Larsen
The Institute for Genomics and Bioinformatics, The Donald Bren School of Information and Computer Sciences, University of California
G. Wesley Hatfield
CODA Genomics, Inc. and University of California
Additional chapters from this book:
Energetics in Escherichia coli-Based Batch Cell-Free Systems
Kalavathy Sitaraman and Deb K. Chatterjee
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 c...
Cell-Free Synthesis of Membrane Proteins for X-Ray Crystallography
Julia E. Fletcher, Federico Katzen, Wieslaw Kudlicki, Yen-Ju Chen, Andrew Chen, Samatha Lieu and Geoffrey Chang
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 selenom...
Cell-Free Expression for Protein NMR
A.J. Shaka
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 enhanc...
Cell-Free Synthesis of Defined Protein Conjugates by Site-Directed Cotranslational Labeling
Michael Gerrits, Jan Strey, Iris Claußnitzer, Uritza von Groll, Frank Schäfer, Martina Rimmele and Wolfgang Stiege
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 studie...
The Role of Cell-Free Rabbit Reticulocyte Expression Systems in Functional Proteomics
Michele Arduengo, Elaine Schenborn and Robin Hurst
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 me...
The PURE system: A Minimal Cell-Free Translation System
Bei-Wen Ying, Yoshihiro Shimizu and Takuya Ueda
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 Escherichi...
Cell-Free Protein Synthesis for Protein Microarrays
Gregory A. Michaud, Michael Salcius, Rebecca Martone, Diane Buhr, John C. Duarte, Jennifer E. McCague, Xiangdong Liu, Michael Samuels, Christine Stalder, James Ball, Alex Tikhonov, Shiranthi Keppetipola, Wieslaw Kudlicki , James Meegan and Barry I. Schweitzer
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...
Bacterial Cell-Free Expression Systems for High-Throughput Protein Production
T.V.S. Murthy, Leonardo Brizuela and Joshua LaBaer
The combination of rapid protein expression systems with high-throughput protein isolation methodologies leads to increased throughput in protein production, improves efficiency and promotes multiplex...
Disulfide Bond Formation in Bacteria-Based Cell-Free Protein Expression
Aaron R. Goerke and James R. Swartz
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 simplif...
Advantages and Applications of the Batch-Formatted E. coli Cell-Free Expression System
Julia E. Fletcher, Federico Katzen, Shiranthi Keppetipola, Ashley Getbehead and Wieslaw Kudlicki
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 c...
Application of in Vitro Virus (IVV) Technique for High-Throughput Analysis of Protein-Protein Interactions
Etsuko Miyamoto-Sato and Hiroshi Yanagawa
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 intere...
C-Terminal Labeling of Proteins Using Fluorescently Conjugated Puromycin Derivatives
Ichiro Tabuchi
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 res...
Cell-Free Protein Expression Screening and Protein Immobilization Using Protein Microarrays
Matthew A. Coleman, Paul Hoeprich, Peter Beernink and Julio A. Camarero
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 bec...
Translation Engineering and Synthetic Biology
David A. Roth, Liza S.Z. Larsen and G. Wesley Hatfield
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 ...
Cell-Free Expression Approaches for the Production and Characterization of Membrane Proteins
Daniel Schwarz, Christian Klammt, Alexander Koglin, Florian Durst, Frank Löhr, Volker Dötsch and Frank Bernhard
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 restric...
Accelerated Protein Evolution Using Ribosome Display
Julie Douthwaite, Lutz Jermutus and Ronald Jackson
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 di...
Cell-Free Protein Expression Labeling with Fluorophores
Jerzy Olejnik
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 ...
Advances in Insect-Based Cell-Free Protein Expression
Uritza von Groll, Stefan Kubick, Helmut Merk, Wolfgang Stiege and Frank Schäfer
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 advan...
