1. Introduction
There is a little doubt that increasing developments of protein synthesis are in high demand. Not only proteins are participants in all biochemical processes of the living cell, continually accelerating advances in proteomics, (i.e. the science of proteins and their reciprocal interactions in the cell) are increasingly underscoring the need to perfect techniques that facilitate the production of specified proteins at an industrial scale that meets the necessary standards of purification (Kim and Kim 2009). Investigations that have built the foundation for such protein production have largely originated from discoveries in the middle of the last century. Such advances firstly elucidated new cellular environments of protein production. Subsequent developments focused on the specificity of protein synthesis and the general efficiency of production has been developed largely by genomic analysis and genetic recombination.
Several
An important criterion involves also simplicity of the system and its potential application: (1) simple systems, such as synthesis of phenylalanine homopolymer (
The most advanced cell-free system based on the application of semi-permeable membrane allowing the concentration of reaction compartment during the work with ribosomes. Such membrane separates the feeding compartment where energy-rich molecules are deposed and can be moved to the reaction compartment with a simple diffusion. Moreover, such a feeding compartment is a suitable space where by-products potentially interfering with the biosynthesis can be deposed.
Recently, many of different cell-free based systems are available and the customer can select the most suitable for the specific application. Here, we described the most popular systems and we demonstrated how these systems can be utilized to study interactions between antibiotics and the ribosome.
1.1. The beginning of cell-free protein synthesis
In 1950s, several research teams independently demonstrated that protein biosynthesis can take place even after disintegration of the cell membrane (Siekievitz and Zamecnik 1951; Borsook et al. 1950; Winnick 1950; Gale and Folkes 1954). Thus, the isolated cytoplasm has been found to comprise the entire set of components necessary to conduct protein biosynthesis. As first, Zamecnik prepared fully active cell-free system based on mitochondrium-isolated ribosomes from an animal (Littlefield et al. 1955; Keller and Littlefield 1957). The team further demonstrated that the reactions were dependent on the supply of high energy molecules, such as ATP and GTP. The first
The discovery of protein expression systems on the template of exogenous mRNA molecules significantly extended applications of extracellular protein biosynthesis. The achievement took place in 1961 in the laboratory of Nirenberg and Matthaei (Nirenberg and Matthaei 1961). A short incubation at the physiological temperature of around 37ºC proved sufficient to remove endogenous mRNA molecules from ribosomes. Free ribosomes obtained from the procedure were subsequently used for protein synthesis on the template of exogenous mRNA molecules. Of great importance, the ribosomes could be "programmed" by synthetic mRNA molecules. The technique of Nirenberg became the classical system of extracellular protein synthesis and, taking advantage of it, its originator deciphered the genetic code, for which he received the Nobel prize in 1968. In the subsequent systems, additional procedures of purifying ribosomes from endogenous mRNA molecules were applied to DEAE cellulose, permitting the separation ribosomes from free nucleic acids
Incubation of ribosomes, preceding the proper protein biosynthesis and conducted in the same manner as in the technique of Nirenberg, was later successfully applied in eukaryotic
1.2. Simple systems based on synthesis of protein homopolymers
In such systems, the principal homopolymeric system involves synthesis of polyalanine on the template of poly(U) chain (
The homopolymeric system was prepared by isolation of two cellular fractions, which were subsequently enriched in high-energy molecules, free amino acides and poly(U)-mRNA, providing the template. The fractions were obtained from bacterial extracts, which were fractionated by centrifugation (for a detailed description of ribosome isolation see (Blaha et al. 2000)). The so-called fraction S30 (obtained by centrifugation at approximately 30,000 rpm for 24 h) was rich in ribosomes and was used to purify free ribosome subunits in a sucrose gradient (centrifugation at around 45,000-60,000 rpm for 15 h). Ribosomes prepared in this manner were incubated at the temperature of 37ºC in a buffer containing, for instance, Mg2+ ions at the concentration of 4.5 mM in order to obtain complete correct 70S ribosome structure capable of performing protein synthesis.
S100 fraction was obtained from supernatant of the S30 fraction and it provides the source of protein factors indispensable to conduct translation (i.a., initiation factors: IF1, IF2, IF3, specific aminoacyl-tRNA synthetases, elongation factors: EF-Tu, EF-G, EF-Tu).
The reaction of polyphenylanaline synthesis represents a simple and widely used technique in several varieties. Instead of a poly(U) template, a poly(A) template can be used, enabling the synthesis of polylysine. Unfortunately, however, the polymer was poorly soluble in water; this property markedly restricts applicability of the system at a broader scale. Nevertheless, application of certain detergents permits its application in studies as seen in previous experiments conducted with the functional analysis of two antibiotics (pactamycin and edein), representing inhibitors of protein synthesis (Dinos et al. 2004). Here, the incorporation of near-cognate lysine instead of phenylalanine on the template of poly(U) can be precisely measured using double radioisotope labeling. If any antibiotic impacts on the translation accuracy (for example aminoglycoside paromomycin) it can be confirmed by detection of higher incorporation of lysine. Followed that technique edein was found to be an error-prone antibiotic in contrast to pactamycin which did not induce any miscoding (Dinos et al. 2004).
1.3. Biosynthesis of protein in a couple transcription-translation system
Nirenberg and Matthaei (Matthaei and Nirenberg 1961) again were the first to describe the DNA dependence of the bacterial extracellular synthesis of protein. The dependence was corroborated by synthesis of a protein on the template of endogenous DNA molecules. Another group of investigators extended synthesis of endogenous proteins by application of exogenous DNA, which originated from a bacteriophage (Byrne et al. 1964; Wood and Berg 1962). Unfortunately, the systems manifested a relatively poor efficiency using either endogenous or viral DNA. Moreover, they were accompanied by a non-specific expression of cellular and bacteriophage proteins. However, the continuing improvements of the joint transcription and translation system resulted in its dissemination; with it ultimately becoming a significant laboratory tool (Lederman and Zubay 1967; DeVries and Zubay 1967).
In the improved system, suggested by Zubay, a preliminary bacterial extract was subjected to incubation in order to degrade mRNA and DNA molecules by cellular nucleases (Zubay 1973). The system gained popularity due to the ease of its preparation, stability of components and a relatively high efficiency. In the system designed by Gold and Schweiger ribosomes were isolated from cellular extracts to their homogenous form and so prepared ribosomes were supplemented with a cytoplasmic fraction, cleared of nucleic acids by ion-exchange chromatography (Schweiger and Gold 1969, 1969, 1970). Such a preparation of components for extracellular protein synthesis produced a remarkable reduction in the non-specific expression of protein. The troublesome procedure, however, remained the disadvantage of the system.
2. Contemporary systems of the cell-free protein expression
The 1980s and 1990s witnessed development of the
The several years of studies on structure and function of individual elements of mRNA sequence resulted in a design of the optimum expression vector for the
2.1. In vitro systems based on application of a semipermeable membrane
At present, the
The RTS is based not only on the ingenious application of a semipermeable membrane but also coupling the transcription and translation reactions, used also in the earlier designed systems (Fig. 1A). Such an approach markedly abbreviated duration of the process and reduced formation of nonspecific products since only the gene present in the expression vector was undergoing transcription and, then, translation.
2.2. Advantages and drawbacks of RTS
The RTS system manifests several advantages. Due to release of ribosomes from the cell and provision of appropriate conditions for the translation reaction, toxic proteins can be produced. If using
The open nature of RTS systems and other
A typical marker protein used in studies on
Using this system, it was possible to demonstrate that the
Aminoglycosides were also tested in another
Nevertheless, extracellular protein biosynthesis is linked to disadvantages which for several years have been successively eliminated. In the course of studies the systems such as RTS was found to support expression of relatively high amounts of protein but around half of the proteins were found to be biologically inactive (Iskakova et al. 2006). However, application of specific translation factors as EF-4 allowed reaching 100% efficiency of RTS system (Qin et al. 2006).
The causes of lowered activity of proteins inside
3. Energy consumption and its regeneration inside in vitro systems
Protein biosynthesis represents a process of particular energetic requirements. In biological systems the energy is obtained from hydrolysis of high energy bonds. For introduction of a single amino acid to the growing polypeptide chain, the cell sacrifices as many as 10 high energy bonds which is equivalent to hydrolysis of 10 molecules of ATP or GTP, each characterized by bonding energy of ΔG0 = -6 kcal/mol. The extreme energetic requirement of a cell supporting protein biosynthesis explains development of sophisticated systems which control energy loss. However, the systems retain the Achilles heel of contemporary systems of protein biosynthesis
Therefore, in the techniques of protein biosynthesis
One of the ways in which the lowered concentration of Mg2+ ions can be avoided involves transformation of orthophosphoric to acetylphosphate using pyruvate oxidase and a defined prosthetic group (TPP or FAD). The reaction requires an access of molecular oxygen, the availability of which is restricted inside
However, the above approach still hardly can be considered ideal. First of all, the restricted access of molecular oxygen markedly reduces the potential for utilization of the system on a larger scale. At present, the solution widely applied involves application of a combined system, based on the traditional PEP/pyruvate kinase approach with acetyl phosphate synthesis by acetyl-CoA, which allows for an effective elimination of free phosphoric acid during synthesis of acetylphosphate from acetyl-CoA (Jewett and Swartz 2004). Application of the system provided a breakthrough and permitted milligram quantities of the produced protein in a volume of just one milliliter (Iskakova et al. 2006).
4. Biotechnological application of extracellular protein synthesis systems
4.1. Efficient expression of several proteins and their screening analysis in parallel
The extracellular protein expression provided the base for many automated techniques of high-throughput expression and screening of several proteins in parallel (Angenendt et al. 2004; Spirin 2004). In such a system, using a single 96- or 384-well plate, multiple genes can be copied, transcribed and translated in parallel, providing substrates for subsequent high-throughput analysis of protein functions. The extensive scale of expression as well as the rate of analysis warrant that the technique deserves to be considered in proteomics and protein engineering based on screening analysis of gene and protein libraries as well as of entire genomes and proteomes (He and Taussig 2007, 2008). The
Such solutions can be proved by the technology known as
The IVEC technique can further be improved by combining it with gene cloning and amplification using the PCR reaction, thus eliminating the time-consuming cloning of the genes to plasmids (Gocke and Yu 2009). Preparation of the appropriate primers containing promoter sequences and Shine-Dalgarno sequences has facilitated protein synthesis directly from products of the PCR reaction (Rungpragayphan, Nakano, and Yamane 2003). The example includes an application of extracellular protein expression system for screening analysis of the entire
Systems of
4.2. Production of proteins ”resistant” to expression
Considering the open character of
4.3. Analysis of molecular interactions
Extracellular protein expression systems markedly facilitate the molecular analysis of interactions between protein and X substance, where X may involve another protein, DNA, RNA or a ligand (Jackson et al. 2004). In order to identify the interaction, one of the reactants must be labeled (a protein, nucleotide or ligand) and placed in a system in which protein, the other reactant is synthesized. Then, the arising complex is isolated from the reaction mix using immunoprecipitation (Derbigny et al. 2000) or it may be directly analyzed in agarose or polyacrylamide gels (Lee and Chang 1995).
4.4. Protein display technologies
The essence of protein display technologies involves establishing a link between genetic information (genotype) and function of an unknown protein (phenotype) in the protein library. The principal technique involves a ribosome display (He and Taussig 1997; Hanes and Pluckthun 1997). Elimination of the STOP codon in mRNA permitted to obtain stable complexes of mRNA-ribosome-protein. Thus, a kind of a frozen structure was obtained, from the threshold of genetic world and proteomics. Subsequently, binding of the protein formed on the ribosome to a defined ligand (which may involve also DNA or RNA) resulted in development of an informational link between a given ligand and the sequence of protein mRNA. Then, a given mRNA-ribosome-protein-ligand complex can be isolated by affinity chromatography from the medium containing also other ligands while mRNA sequences are identified by reverse transcription and DNA sequencing. In order to amplify efficacy of the system, the process is conducted in a cyclic manner, i.e., the isolated mRNAs are independently amplified and added again to the mixture of ribosomes and ligands, enabling a more effective selection of an individual specific ligand. In combination with methods of genetic engineering, including mutagenesis, the protein display technologies can be applied not only in proteomics but also in molecular evolution studies. Now, the processes of interactions between DNA, RNA and protein, which took millions of years of evolution may be analyzed in the laboratory and their rate may be multiplied by selective amplification of DNA.
5. Conclusion
Cell-free systems will be optimized and improved according to their expression yield, protein specificity (“difficult proteins”) and protein folding. They will be more broadly applied in protein microarrays technology where can be utilized for the analysis of protein-protein interaction. Furthermore, protein technologies based on cell-free biosynthesis will be applied for protein engineering in order to synthesis specific antibodies or enzymes, as well as for production of proteins for crystallisation.
The “post-genome” research requires comprehensive tool which will allow determination of structure, function and specific location of the proteins in the network of proteomes. It has to be performed effectively, quickly and on the multiple platform where large number of proteins can be analyzed in the same time. Based on cell-free systems such analysis is possible especially in the comparison to traditional cell-based systems where their miniaturization is rather impossible.
Acknowledgments
This study was supported by the Polish Ministry of Science and Higher Education (grants no. 0172/B/P01/2009/36). Authors thank to Dr. Jan Jaroszewski for his help in lingual edition of the manuscript. Witold Szaflarski thanks to Prof. Knud H. Nierhaus for his long-time powerful mentoring.
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