Towards the construction of expressed proteomes using a Leishmania tarentolae based cell-free expression system

The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed.     

Bacterial cell-free system for high-throughput protein expression and a comparative analysis of Escherichia coli cell-free and whole cell expression systems

Sixty-three proteins of Pseudomonas aeruginosa in the size range of 18-159 kDa were tested for expression in a bacterial cell-free system. Fifty-one of the 63 proteins could be expressed and partially purified under denaturing conditions. Most of the expressed proteins showed yields greater than 500 ng after a single affinity purification step from 50 microl in vitro protein synthesis reactions. The in vitro protein expression plus purification in a 96-well format and analysis of the proteins by SDS-PAGE were performed by one person in 4 h. A comparison of in vitro and in vivo expression suggests that despite lower yields and less pure protein preparations, bacterial in vitro protein expression coupled with single-step affinity purification offers a rapid, efficient alternative for the high-throughput screening of clones for protein expression and solubility.     

A novel cell-free protein synthesis system

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30–45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications.     

Wheat germ systems for cell-free protein expression

Cell-free protein expression plays an important role in biochemical research. However, only recent developments led to new methods to rapidly synthesize preparative amounts of protein that make cell-free protein expression an attractive alternative to cell-based methods. In particular the wheat germ system provides the highest translation efficiency among eukaryotic cell-free protein expression approaches and has a very high success rate for the expression of soluble proteins of good quality. As an open in vitro method, the wheat germ system is a preferable choice for many applications in protein research including options for protein labeling and the expression of difficult-to-express proteins like membrane proteins and multiple protein complexes. Here I describe wheat germ cell-free protein expression systems and give examples how they have been used in genome-wide expression studies, preparation of labeled proteins for structural genomics and protein mass spectroscopy, automated protein synthesis, and screening of enzymatic activities. Future directions for the use of cell-free expression methods are discussed.     

Simultaneous expression and maturation of the iron-sulfur protein ferredoxin in a cell-free system

The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.     

Synaptosomal protein synthesis in a cell-free system

—Synaptosomes were isolated from cerebral cortex of young rats and incubated with ¹⁴C-labelled l -leucine in vitro. Amino acid incorporation into proteins of the synaptosomal cytoplasm, mitochondria and membrane components was observed. There was no incorporation into proteins of the vesicles. The protein-synthesizing system was not stimulated by the addition of either ATP or an ATP-generating system. ATP at all concentrations was inhibitory. Two different protein-synthesizing systems operate in the synaptosome. One, sensitive to inhibition by chloramphenicol and related antibiotics, is found in the mitochondrial subfraction and the other, inhibited by cycloheximide, is located either in the membrane components or the synaptosomal cytoplasm. This second system resembles the eukaryotic ribosomal system in its sensitivity to cycloheximide. Both the synaptosomal soluble fraction and the synaptosomal membrane fractions were shown previously to contain RNA. This RNA could function in protein-synthesizing mechanisms in the synaptosome. These results deomonstrate that protein is synthesized in axonal components and show that it is unnecessary to postulate that all axonal protein is supplied by somato-axonal flow.     

High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system

Cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we review our recent advances in a cell-free protein synthesis system prepared from wheat embryos. We first addressed and resolved the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins. We found that conventional wheat germ extracts contained the RNA N-glycosidase tritin and other inhibitors such as thionin, ribonucleases, deoxyribonucleases, and proteases that originate from the endosperm and inhibit translation. Extensive washing of wheat embryos to eliminate endosperm contaminants has resulted in extracts with a high degree of stability and activity. To maximize the translation yield and throughput of the system, we then focused on developing the following issues: optimization of the ORF flanking regions, a new strategy to construct PCR-generated DNAs for screening, and design of an expression vector for large-scale protein production. The resulting system achieves high-throughput expression, with a PCR-directed system at least 50 genes that can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Under the dialysis mode of reaction, the system with the expression vector can maintain productive translation for 14 days. The cell-free system described here bypasses most of the biological processes and lends itself to robotic automation for high-throughput expression of genetic information, thus opening up many possibilities in the post-genome era.     

High-throughput,genome-scale protein production method based on the wheat germ cell-free expression system

Current cell-free protein expression systems are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. Escherichia coli based systems are not always sufficient for expression of eukaryotic proteins. This report reviews a high-throughput protein production method based on the cell-free system prepared from eukaryote, wheat embryos. We first demonstrate a method for preparation of this extract that exhibited a high degree of stability and activity. To maximize translation yield and throughput, we address and resolve the following issues: (1) optimization of the ORF flanking regions; (2) PCR-based generation of DNA for mRNA production; (3) expression vectors for large-scale protein production; and (4) a translation reaction that does not require a membrane. The combination of these elemental processes with robotic automation resulted in high-throughput protein synthesis.     

Screening for soluble expression constructs using cell-free protein synthesis

The SH2 domain of STAT6 was chosen to test the in vitro protein synthesis as a screening tool. Goal of the screening was to obtain constructs which produce soluble protein in E. coli. The expression of 70 different constructs using an E. coli based cell-free system revealed two constructs, which give partly soluble protein. The introduction of two mutations, which had been suggested by a structural based alignment of 20 different SH2 domains lead to increased solubility. The expression of both constructs in E. coli followed by an affinity and size exclusion chromatography resulted in milligram quantities of highly purified protein.     

Statistical analysis of features associated with protein expression/solubility in an in vivo Escherichia coli expression system and a wheat germ cell-free expression system

Recombinant protein technology is an important tool in many industrial and pharmacological applications. Although the success rate of obtaining soluble proteins is relatively low, knowledge of protein expression/solubility under 'standard' conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we conducted a genome-scale experiment to assess the overexpression and the solubility of human full-length cDNA in an in vivo Escherichia coli expression system and a wheat germ cell-free expression system. We evaluated the influences of sequence and structural features on protein expression/solubility in each system and estimated a minimal set of features associated with them. A comparison of the feature sets related to protein expression/solubility in the in vivo Escherichia coli expression system revealed that the structural information was strongly associated with protein expression, rather than protein solubility. Moreover, a significant difference was found in the number of features associated with protein solubility in the two expression systems.     

Enhanced cell-free protein expression by fusion with immunoglobulin Ckappa domain

While cell-free systems are increasingly used for protein expression in structural and functional studies, several proteins are difficult to express or expressed only at low levels in cell-free lysates. Here, we report that fusion of the human immunoglobulin kappa light chain constant domain (Ckappa) at the C terminus of four representative proteins dramatically improved their production in the Escherichia coli S30 system, suggesting that enhancement of cell-free protein expression by Ckappa fusion will be widely applicable.     

Cloning, expression, and identification of a novel class IIa bacteriocin in the Escherichia coli cell-free protein expression system

The NB-C1 gene, acquired from the result of data mining of the lactic acid bacteria genome, is a novel potential class IIa bacteriocin gene with the characteristic YGNGVxC cluster. To produce soluble NB-C1 efficiently and overcome issues of protein toxicity, we adopted a GFP fusion strategy using an Escherichia coli cell-free protein expression system. We constructed the expression vector pIVEX2.4d-GFP-NB-C1, which was expressed in both the batch mode and the continuous exchange cell-free (CECF) systems. The amount of soluble fusion protein achieved from the CECF system (2.2 mg/ml) was approximately three times higher than that in the batch mode (0.73 mg/ml). The soluble fusion protein was purified via one-step Ni–NTA affinity chromatography, with a concentration of 0.26 mg/ml and a purity of 95%. The purified NB-C1 showed strong antimicrobial activity against the indicator bacteria Listeria monocytogenes.     

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system

Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components.     

Tuning the expression level of recombinant proteins by modulating mRNA stability in a cell-free protein synthesis system

In this work, it was discovered that the stability of mRNA in a cell-free extract could be controlled by using engineered T7 terminator sequences. Specifically, it was found that mRNA stability gradually decreased as the length of the stem structure of the T7 terminator was reduced sequentially. As a result of the controlled abundance of mRNA species, it was possible to manipulate the relative expression level of target proteins by employing the T7 terminator of adjusted stem lengths.