Cell-free Protein Synthesis

Methods and Protocols

Alexander S. Spirin, James R. Swartz

• Publisher: Wiley-VCH
• Year: 2014
• Print ISBN: 9783527316496
• eBook ISBN: 9783527691500
• Edition: 1
• Topic: Biowissenschaften
• Subtopic: Biochemie

With its detailed description of membrane protein expression, high-throughput and genomic-scale expression studies, both on the analytical and the preparative scale, this book covers the latest advances in the field. The step-by-step protocols and practical examples given for each method constitute practical advice for beginners and experts alike.

1

Cell-free Protein Synthesis Systems: Historical Landmarks, Classification, and General Methods

Alexander S. Spirin and James R. Swartz

1.1 Introduction: Historical Landmarks

1.1.1 Discovery of Protein Synthesis in Cell Extracts

The demonstration of the capability of disintegrated cells and cell extracts to continue protein synthesis was among the great discoveries of the early 1950s that led to the birth of molecular biology. The original observations were made independently in several laboratories working with homogenates and homogenate fractions of animal tissues [16, 132, 154, 155, 174, 175]. Shortly afterwards, disrupted bacterial cells were also shown to be capable of synthesizing proteins [42] and the fraction of ribonucleoprotein particles called ribosomes was identified as the heart of the protein-synthesizing machinery of the cell [138]. Cell extracts freed from heavy components by centrifugation at 30000g (the so-called S30 extracts) and supplemented with amino acids, ATP and GTP, were the first cell-free protein-synthesizing systems [65, 95, 99, 100, 114, 137, 148, 168].

In those systems, however, the ribosomes just continued to translate endogenous mRNAs and elongate the polypeptides for which synthesis had already been started. Nevertheless, a high level of globin synthesis from endogenous mRNA templates could be achieved using rabbit reticulocyte lysates, and several general molecular mechanisms of protein-synthesizing machinery were studied (see, e.g., Refs. [54, 135]).

1.1.2 Translation of Exogenous Messages

The principal breakthrough in the development of cell-free protein-synthesizing systems was made in 1961 when Nirenberg and Matthaei managed to destroy endogenous mRNA in the bacterial (E. coli) extract without damaging ribosomes and the rest of the protein-synthesizing machinery [124, 125]. The ribosome run-off and the selective destruction of endogenous mRNAs was accomplished by a simple procedure of pre-incubation of the extract at 30–37 °C. The introduction of polyribonucleotides in such mRNA-depleted extracts resulted in effective translation of exogenous messages. The experiments with translation of synthetic polyribonucleotides, such as poly(U), poly(A), poly(C) and random copolymers poly(U,C), poly(U,C,A), etc., deciphered the genetic code. Furthermore, natural alien messages, including eukaryotic mRNAs, could be successfully expressed in such bacterial extracts.

Later, addition of a Ca²⁺-dependent micrococcal ribonuclease with subsequent inactivation of the enzyme by removal of Ca²⁺ with EGTA was used for destruction of endogenous globin mRNA in the reticulocyte lysate [130]. Such mRNA-depleted animal extracts became the basis of the most efficient eukaryotic cell-free systems [55, 57, 115].

Another type of eukaryotic cell-free system was based on wheat germ extracts [106, 136]. In this case the content of active endogenous mRNA in the extract was found to be so low that there was no need for the pre-treatment procedures described above [5, 105].

1.1.3 Coupled Transcription-translation in Bacterial Extracts

In the case of bacterial cell-free translation systems, the addition of pre-synthesized mRNA to a cell extract violates the principle of natural prokaryotic translation. In prokaryotic cells, translation of a mRNA by ribosomes is initiated soon after the beginning of its synthesis on the DNA template. The ribosomes move along mRNA chain not far behind the RNA polymerase, and both processes proceed with synchronized rates (coupled transcription-translation) [102, 181]. When pre-synthesized, complete mRNAs are used in cell-free translation systems, the initiation of translation sometimes may be hindered by mRNA folding and tertiary structure formation, especially if ribosome-binding sites are involved.

The first demonstration of DNA-dependent incorporation of amino acids into synthesized proteins as well as the first evidence for the coupled transcription-translation process in bacterial cell-free systems were also made by Nirenberg’s group in the beginning of the 1960s [21, 110]. In 1967, Zubay and colleagues made significant improvements [36, 97] and introduced an efficient bacterial coupled transcription-translation system for expression of exogenous DNA [27, 189]. Their system was based on crude cell-free E. coli extract containing endogenous RNA polymerase, but devoid of endogenous DNA and mRNA due to exhaustive nuclease degradation. This improved method was broadly adopted, although somewhat modified protocols were reported by others (see, e.g., Ref. [134]).

Another practical version of the bacterial coupled transcription-translation system for exogenous gene expression was proposed by Gold and Schweiger [49, 50, 149], who used the mixture of isolated E. coli ribosomes, tRNA and ribosome-free supernatant (the so-called S100 extract) purified by ion-exchange chromatography from all nucleic acids, instead of the crude extract with degraded nucleic acids.

1.1.4 Combined Transcription-translation Systems

The next important step in the development of cell-free gene expression was the combination of a cell extract with a specific bacteriophage RNA polymerase that used a phage-specific promoter for transcription. Either SP6 polymerase [161] or T7 polymerase [30, 123] were suggested for such cell-free systems. These polymerases direct the exclusive synthesis of the proteins encoded by genes preceded by the corresponding phage promoters. Such systems possess several advantages: (a) the phage polymerases provide a higher level of transcripts than endogenous bacterial RNA polymerase; (b) the addition of rifampicin selectively inhibits the endogenous RNA polymerase, and thus there is no need to self-digest or treat cell extract for removal of endogenous DNA; (c) due to the promoter specificity of the phage RNA polymerase, only the gene of interest is expressed; (d) the systems are convenient for expression from any plasmid constructs and PCR products where the simple phage promoters are inserted; and (e) the phage RNA polymerases and DNA templates with phage promoters can be combined both with prokaryotic [123, 161] and eukaryotic [30, 161] extracts.

Strictly speaking, these systems cannot be referred to as “coupled transcription-translation” systems: both spatial and temporal coupling is absent in this case because the T7 and SP6 RNA polymerases work much faster than the endogenous bacterial polymerase and translation machinery, the transcripts quickly accumulate in excess over translating ribosomes, and thus mRNA is synthesized mainly in advance in such systems. The term “combined transcription-translation” is a more appropriate designation. It should be mentioned that both purified E. coli RNA polymerase [18, 28, 98, 137, 164] and animal virus-associated RNA polymerases [7, 29, 131] were also used in the combined cell-free transcription-translation systems based on eukaryotic extracts, but the use of SP6 and T7 phage polymerases proved to be the most successfull.

1.1.5 Continuous Flow/Continuous Exchange Principle

In cell-free translation and transcription-translation systems performed in a fixed volume of a test-tube (batch format) the reaction conditions change during incubation as a result of the consumption of substrates and the accumulation of products. Translation stops as soon as any essential substrate is exhausted or any product or by-product reaches an inhibiting concentration, usually after 20–60 minutes of incubation. The limited lifetimes and, as a consequence, low yields of protein products made the early batch systems useful mainly for analytical purposes and inappropriate for preparative synthesis of polypeptides and proteins.

A principal breakthrough was the invention of the so-called continuous-action or continuous-duty translation [4, 160] and transcription-translation systems [8, 10] (see also Refs. [14, 15, 153, 161, 162]). Instead of incubating the reaction mixture in a fixed volume in a test-tube, the reaction was performed under conditions of persistent supply of the consumable substrates (amino acids, nucleoside triphosphates and energy-regenerating compounds) and with removal of the reaction products (mainly inorganic phosphates and nucleoside monophosphates, as well as polypeptide products and by-products in some reactor versions). To achieve that, a porous (ultrafiltration or dialysis) membrane was used to retain the high-molecular-weight components of the protein-synthesizing machinery (ribosomes, mRNA, ARSases, etc.) within a defined reaction compartment. The membrane separated the reaction compartment from another compartment containing a feeding solution with a reservoir of low-molecular-weight components (substrates) for the reaction. This technique was reproduced in a number of laboratories [37, 38, 66, 90, 126, 127, 171, 172, 177].

In so-called continuous-flow cell-free (CFCF) systems [8, 10, 160] the feeding solution with substrates is continuously pumped into the chamber containing the reaction mixture, and the products are continuously removed through the ultrafiltration membrane by the outgoing flow. Reactors working in pulsating [161] (see also Ref. [162]) or “discontinuous” (see Chapter 7 of this book) modes, with alternating flow-in and flow-out instead of the direct continuous flow, can also be used.

In the dialysis format of the continuous systems, designated also as continuous-exchange cell-free (CECF) systems, the passive (diffusional) exchange of substrates and low-molecular-weight products through a porous barrier takes place [4, 26, 107, 153, 162]. The dialysis (CECF) format was found to be much simpler and more practical than the CFCF format, and became the most widely exploited type of the continuous-action cell-free systems in laboratories (see Refs. [67, 70, 79, 103, 145] and Chapters 5–9), as well as in commercialized technologies (see, e.g., Refs. [13, 33, 34, 108]). Both simple dialysis bags and reactors with flat dialysis membranes are used. The reactors with hollow fibers were also proposed [161, 177]. Notably, the same continuous-exchange principle can be realized also without a dialysis membrane. Reactors have been proposed where the diffusional product/substrate exchange is accomplished between gel capsules that hold the protein-synthesizing mixture and the outside feeding solution [161]. Other formats include the use of Sephadex granules to retain the feeding solution while the reaction mixture occupies the inter-granule space [17], and a configuration in which the reaction mixture and feeding solution exist in two liquid layers separated only by a phase boundary [146] (see also Ref. Chapter 7).

The use of the continuous-action principle in cell-free translation and transcription-translation systems maintains more or less constant reaction conditions and prolongs the active working time of the systems up to many hours or even days. As a result, the yield of the product increased to milligrams of protein per mL of incubation mixture.

1.2 Prokaryotic and Eukaryotic Types of Cell-free Expression Systems

Although the general mechanisms of protein biosynthesis are considered to be universal in the biological world, there are essential differences in initiation of translation and its regulation between prokaryotic (to be more exact, eubacterial) and eukaryotic organisms [159]. Correspondingly, cell-free translation (and transcription-translation) systems based on either eubacterial or eukaryotic extracts are characterized by specific features in their compositions, requirements and in their fields of application. Presently, cell-free systems employing Escherichia coli extracts, on one hand, and wheat germ and rabbit reticulocyte extracts, on the other, are almost exclusively used as standards for in vitro translation systems of the prokaryotic and eukaryotic types, respectively.

1.2.1 Cell Extracts

1.2.1.1 E. coli extract (ECE)

By now the use of E. coli extracts (ECE) for cell-free translation and transcription-translation seems to be the most practical and efficient for in vitro synthesis of functional proteins of both prokaryotic and eukaryotic origin. Detailed knowledge of the protein-synthesizing machinery of the E. coli cells, including structure, genetics, metabolism and regulation of its components and reactions, has enabled remarkable improvements of the bacterial cell-free systems during recent years. Currently, up to 1–1.5 mg mL^–1 of protein (see Chapter 12). It can be produced in an optimized batch cell-free system, and the productivity can be further enhanced up to 10 mg mL^–1 of protein by the use of continuous-action reactors (see Chapters 5 and 7). Although the issue of proper protein folding remains a substantial challenge, especially for the synthesis of multi-domain and disulfide-bonded eukaryotic proteins, virtually any genetic information can be translated into a polypeptide in the ECE systems. Protein synthesis in the ECE systems has been shown to be quite tolerant to various additives, including cofactors, metabolites, unnatural amino acids, and even detergents. This implies that one can modify synthesis and folding conditions to maximize the yield of soluble and functionally active proteins. For example, reaction conditions can be modified to provide an oxidizing environment for the synthesis and folding of proteins containing multiple disulfide bonds. Certain natural modifications of synthesized proteins, such as specific phosphorylation of serine or threonine residues, can be made by addition to the system of modifying enzymes and their substrates. Co-translational and post-translational glycosylation and other complex modifications of eukaryotic proteins, however, are problematic in ECE cell-free systems.

Sometimes serious problems arise with side activities of the extracts, resulting in damage to ribosomes; degradation of plasmids, mRNA, and tRNA; uncoupled hydrolysis of NTPs; and metabolic consumption of certain amino acids. More recently, several important improvements were proposed for extract preparation. One of them is the introduction of additional amounts of some amino acids into a cell-free system (see, e.g., Ref. [73]). In particular, it was shown that arginine, cysteine, serine and tryptophan, and also methionine, aspartic acid, and glutamic acid may be subjected to especially intensive degradation in ECE, and their re-addition is sufficient to stimulate or restore the ECE system activity [72, 77]. The presence of NAD, CoA, and oxalate was shown to be useful for maintenance of energy supply sub-systems (see below) in the ECE cell-free system. The use of a condensed ECE was reported to improve the productivity of the system [67, 73]. The extracts from mutant E. coli strains with reduced degradative activities were also successfully used to increase the lifetime and productivity of the cell-free system [116]. Chapters 3, 4, and 12 describe several of these improvements.

1.2.1.2 Wheat Germ Extract (WGE)

Translation systems based on WGE have been widely used for the syntheses of radioactively labeled polypeptides and proteins, mostly for analytical purposes. Only recently has substantial progress in productivity been achieved for WGE cell-free systems, mainly by extending the duration of the synthesis time (lifetime of the system) (see Chapter 7). Removal of enzymes causing damage to ribosomes, mRNAs, and other components of the protein-synthesizing machinery during extract preparation is the key to keep protein synthesis running up to several days. In this way, the productivity of the WGE systems can be enhanced up to 1 mg mL^–1 of product over 24 hours. The use of continuous-action reactors with continual supply of substrates further increases the duration of the synthesis up to several days, or even 1–2 weeks. Protein synthesis in WGE can be directed by either linear or circular DNA, as well as by either capped or uncapped mRNA. The WGE systems are considered to be more adapted to euka...

Table of contents

• Cover
• Contents
• Title Page
• Copyright
• Preface
• List of Contributors
• 1 Cell-free Protein Synthesis Systems: Historical Landmarks, Classification, and General Methods
• 2 The Constructive Approach for Cell-free Translation
• 3 Functional Genomic Analysis using Sequential Cell-free Protein Synthesis
• 4 Cell-free Technology for Rapid Production of Patient-specific Fusion Protein Vaccines
• 5 Bacterial Cell-free System for Highly Efficient Protein Synthesis
• 6 The Use of the Escherichia coli Cell-free Protein Synthesis for Structural Biology and Structural Proteomics
• 7 The Wheat Germ Cell-free Protein Synthesis System
• 8 Cell-free Expression of Integral Membrane Proteins for Structural Studies
• 9 Cell-free Production of Membrane Proteins in the Presence of Detergents
• 10 Novel Techniques using PCR and Cell-free Protein Synthesis Systems for Combinatorial Bioengineering
• 11 Gene Cloning and Expression in Molecular Colonies
• 12 Large-Scale Batch Reactions for Cell-free Protein Synthesis
• Index