Cell-free Protein Expression Research Articles

Nanoscale Pipetting for Controlled Chemistry in Small Arrayed Water Droplets Using a Double-Barrel Pipet

We present a new methodology which provides for the miniaturization of one of the most common tools in use in chemistry and biology laboratories today-the micropipet. We have used glass-fabricated double-barrel nanopipets to controllably produce arrayed water droplets with volumes as small as a few attoliters under an organic layer. We have addressed individual droplets and added controlled amounts of either additional volume or reagents from one of the barrels of the pipet. We demonstrate that this method can be used for miniaturized cell-free protein expression.

Combination of cell-free expression and NMR spectroscopy as a new approach for structural investigation of membrane proteins

Despite major technical advance in methods used for structural investigations of proteins structure determination of membrane proteins still poses a significant challenge. Recently, the application of cell-free expression systems to membrane proteins has demonstrated that this technique can be used to produce quantities sufficient for structural investigations for many different membrane proteins. In particular for NMR spectroscopy, cell-free expression provides major advantages since it allows for amino acid type selective and even amino acid position specific labeling. In this mini-review we discuss the combination of cell-free membrane protein expression and liquid state NMR spectroscopy.

Cell-Free Protein Synthesis Containing Disulfide Bonds and Its Application to Protein Engineering

The cell-free protein synthesis system, which is sometimes called in vitro expression, has been remarkably improved in the past decade. However, proteins containing disulfide bonds could not been produced as active forms using the conventional cell-free system because it was carried out under reduced conditions. We have demonstrated that modification of the reaction conditions, i.e., no addition of reducing reagent, removal of the reducing reagent from the E. coli S30 extract by dialysis, and the addition of some molecular chaperons, enabled the correct folding of such proteins as Burkholderia cepacia lipase, Streptomyces antibioticus phospholipase D, Phanerochaete chrysosporium manganese peroxide (MnP), and artificial antibodies (scFv and Fab). As an application of cell-free protein synthesis, we have developed a novel high-throughput protein library construction system by combining the amplification of a single DNA molecule by means of PCR with a cell-free protein expression system. This completely cell-free system is termed SIMPLEX (single-molecule-PCR-linked in vitro expression). By applying SIMPLEX, we have achieved (1) inversion of the enatioselectivity of the bacterial lipase, (2) improvement in the H2O2 stability of the fungal MnP, and (3) enhancement of the binding affinity of anti-human-serum-albumin scFv.

Translational incorporation of L‐3,4‐dihydroxyphenylalanine into proteins

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean 15N-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors

In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1 h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1 mg/ml of reaction volume for 6–8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.

ここまできたプロテオミクス研究技術とその応用 ラピッドトランスレーションシステム

The post-genomic era brings with it many challenges to the understanding of protein structure, function and interaction. The increasing number of newly identified proteins that need to be expressed, problems associated with cell cultivation, and traditional sub-cloning procedures are some of the key bottlenecks.Rapid Translation System “RTS” is a scalable in vitro transcription/translation protein expression platform, that produces sufficient amounts of protein for characterization studies, functional assays, or structural analysis. RTS offers the advantages of a cell-free protein expression system, eliminating the need for laborious up- and down-stream steps (e.g. host cell transformation, culturing, or lysis) typically associated with cell-based expression systems.Generally, to further fasten the protein expression screening, we recommend to use linear (PCR-generated) templates with the RTS screening kits. These templates are produced via the RTS Linear Template Generation Sets. For scale-up with RTS500 (1ml reaction) and RTS9000 (10, 30ml reaction), the use of circular templates is recommended, which are generated with the RTS pIVEX Vector Sets. In order to yield mg quantities, expressions should be performed using the RTS ProteoMaster instrument.For optimization of the prokaryotic Rapid Translation, we offer a special software, the ProteoExpert RTS E. coli HY. The software delivers high-yield variants of an input ORF sequence.

Towards the high-throughput expression of metalloproteins from theMycobacterium tuberculosisgenome

The provision of high-quality protein in adequate quantities is a prerequisite for any structural genomics programme. A number of proteins from the Mycobacterium tuberculosis genome have been expressed and the success at each stage of the process assessed. Major difficulties have been encountered in the purification and solubilization of many of these proteins, most likely as a result of mis-folding. Some improvements have been made to the protocol but the overall success rate is still limited; however, the use of a cell-free protein expression system will circumvent some of the difficulties encountered. Alternative purification systems are also required and the properties of a mutant blue copper protein are described, that may offer a combined purification and tagging system.

Antisense Phosphorodiamidate Morpholino Oligomer Length and Target Position Effects on Gene-Specific Inhibition inEscherichia coli

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic DNA analogs that inhibit gene expression in a sequence-dependent manner. PMOs of various lengths (7 to 20 bases) were tested for inhibition of luciferase expression in Escherichia coli. Shorter PMOs generally inhibited luciferase greater than longer PMOs. Conversely, in bacterial cell-free protein synthesis reactions, longer PMOs inhibited equally or more than shorter PMOs. Overlapping, isometric (10-base) PMOs complementary to the region around the start codon of luciferase inhibited to different extents in bacterial cell-free protein expression reactions. Including the anti-start codon in PMOs was not required for maximal inhibition. PMOs targeted to 5' nontranslated or 3' coding regions within luciferase mRNA did not inhibit, except for one PMO targeted to the ribosome-binding site. Inhibition of luciferase expression correlated negatively with the predicted secondary structure of mRNA regions targeted by PMO but did not correlate with C+G content of targeted regions. The effects of PMO length and position were corroborated by using PMOs (6 to 20 bases) targeted to acpP, a gene required for viability. Because inhibition by PMOs of approximately 11 bases was unexpected based on previous results in eukaryotes, we tested an 11-base PMO in HeLa cells and reticulocyte cell-free protein synthesis reactions. The 11-base PMO significantly inhibited luciferase expression in HeLa cells, although less than did a 20-base PMO. In reticulocyte cell-free reactions, there was a trend toward more inhibition with longer PMOs. These studies indicate that strategies for designing PMOs are substantially different for prokaryotic than eukaryotic targets.

A Novel Method for Producing Custom-Made Idiotype Vaccines for Lymphoma Immunotherapy Using a Cell-Free Expression System.

Idiotype vaccines have been used as a customized immunotherapy for lymphoma. This strategy takes advantage of a unique antigen (either immunoglobulin or T cell receptor) found on the lymphoma cells of each patient. To make this personalized vaccine feasible for widespread use a production method, which is rapid and relatively inexpensive will be required. In an E. coli-based cell-free protein expression system we were able to make ScFv proteins derived from the immunoglobulins of a mouse B cell lymphoma, 38C13, in high yields. We have used this cell-free system to make 38C13 ScFv proteins fused to either the cytokine mGM-CSF or a 9 amino acid peptide derived from hIL-1b. The 9 amino acid fragment of hIL-1b was chosen because it is known to retain immunostimulatory function while lacking pyrogenicity. We were able consistently to produce these proteins in high yield and to purify them using a histadine tag at the terminus of the molecule. The ScFv portion of the molecule is folded properly, as assayed by its binding to an anti-idiotype Ab. Similarly, the mGM-CSF fused to the ScFv can be recognized by an anti-mGM-CSF Ab. The mGM-CSF portion is biologically active as determined by its ability to support the growth a mGM-CSF dependant cell line. We immunized C3H mice with either the ScFc-hIL-1b or the ScFv-mGM-CSF fusion protein and we were able to induce an idiotype-specific Ab response, which recognizes the native idiotype protein in ELISA assays as well as the 38C13 tumor as detected by flow cytometry. Most importantly, in the highly aggressive 38C13 lymphoma model system, three bi-weekly vaccinations with either fusion protein protected mice from tumor challenge with the syngeneic 38C13 tumor. These data establish that a cell-free protein expression system can work for producing individualized idiotype vaccines for lymphoma. (This work was supported by NIH grant CA34233)

Expression and activity of the cytolethal distending toxin of Helicobacter hepaticus*1

Helicobacter hepaticus , a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.

Genome-scale, biochemical annotation method based on the wheat germ cell-free protein synthesis system

Since the complete genomic DNA sequencing of various species, attention has turned to the structural properties, and functional characteristics of proteins. Current cell-free protein expression systems from eukaryotes are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. This report reviews the high-throughput, genome-scale biochemical annotation method based on the cell-free system prepared from wheat embryos. We first briefly reviewed our highly efficient and robust wheat germ cell-free protein synthesis system, and then showed an application of the system for materialization and characterization of genetic information taking a cDNA library of protein kinase from Arabidopsis thaliana as an example. The procedure consists of: (1) fusion of the gene-of-interest to a purification-tag, amplified by the split-primer PCR method; (2) transcription and purification of mRNA; (3) cell-free protein synthesis in the bilayer system using 96-well titer plate; (4) affinity purification and activity measurement. We took 439 cDNAs encoding kinases among 1064 genes annotated so far, and they were translated in parallel into protein. Subsequent assay revealed 207 products having autophosphorylation activity. Furthermore, seven proteins out of 26 calcium-dependent protein kinase genes tested did phosphorylate a synthetic peptide substrate in the presence of calcium ion, demonstrating that the translation products, retained their substrate specificity. The information on biochemical function of gene products accumulated should revolutionize our understanding of biology and fundamentally alter the practice of medicine and influence other industries as well.

Expression and activity of the cytolethal distending toxin of Helicobacter hepaticus

Helicobacter hepaticus, a causal agent of hepatocarcinoma in mice, exhibits a cytolethal distending toxin activity. The three subunits of this holotoxin, CdtA, CdtB, and CdtC, and three CdtB mutants were produced as recombinant histidine-tagged proteins by using an in vitro cell-free protein expression system. We found that the presence of the three H. hepaticus Cdt subunits is required for cellular toxicity and that only a C-terminal CdtB mutation abolishes the activity of the complex. In vitro, H. hepaticus CdtB exhibits a DNase activity which is also abolished by this C-terminal CdtB mutation. These results suggest that the effect of H. hepaticus CDT probably involves the DNase activity of CdtB.

Genome-scale, biochemical annotation method based on the wheat germ cell-free protein synthesis system

Since the complete genomic DNA sequencing of various species, attention has turned to the structural properties, and functional characteristics of proteins. Current cell-free protein expression systems from eukaryotes are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. This report reviews the high-throughput, genome-scale biochemical annotation method based on the cell-free system prepared from wheat embryos. We first briefly reviewed our highly efficient and robust wheat germ cell-free protein synthesis system, and then showed an application of the system for materialization and characterization of genetic information taking a cDNA library of protein kinase from Arabidopsis thaliana as an example. The procedure consists of: (1) fusion of the gene-of-interest to a purification-tag, amplified by the split-primer PCR method; (2) transcription and purification of mRNA; (3) cell-free protein synthesis in the bilayer system using 96-well titer plate; (4) affinity purification and activity measurement. We took 439 cDNAs encoding kinases among 1064 genes annotated so far, and they were translated in parallel into protein. Subsequent assay revealed 207 products having autophosphorylation activity. Furthermore, seven proteins out of 26 calcium-dependent protein kinase genes tested did phosphorylate a synthetic peptide substrate in the presence of calcium ion, demonstrating that the translation products, retained their substrate specificity. The information on biochemical function of gene products accumulated should revolutionize our understanding of biology and fundamentally alter the practice of medicine and influence other industries as well.

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.

Preparation of Escherichia coli cell extract for highly productive cell-free protein expression.

As structural genomics and proteomics research has become popular, the importance of cell-free protein synthesis systems has been realized for high-throughput expression. Our group has established a high-throughput pipeline for protein sample preparation for structural genomics and proteomics by using cell-free protein synthesis. Among the many procedures for cell-free protein synthesis, the preparation of the cell extract is a crucial step to establish a highly efficient and reproducible workflow. In this article, we describe a detailed protocol for E. coli cell extract preparation for cell-free protein synthesis, which we have developed and routinely use. The cell extract prepared according to this protocol is used for many of our cell-free synthesis applications, including high-throughput protein expression using PCR-amplified templates and large-scale protein production for structure determinations.

Semisynthesis of hemoglobin.

Protein engineering, generation of mutant or modified forms of protein, has become the first step for studying the correlation of structure and function of proteins. Design and generation of novel protein molecules with tailormade properties is the long-range goal of such studies. Such novel protein molecules are now designed and generated by recombinant DNA technology, as long as these protein molecules contain only the naturally occurring 20 amino acids residues (,). However, if unnatural amino acids needs to be introduced, cell-free protein expression system and special manipulation of the tRNA is necessary (,). Incorporation of the unnatural amino acid residues into a protein in a site-specific fashion could also be achieved through total chemical synthesis. Gutte and Merrifield () achieved the total chemical synthesis of RNase-A using solid-phase synthesis, starting from the carboxyl end of the molecule and building, one residue at a time, to its amino terminus. Hoffman et al. () introduced an alternate approach that involved the synthesis of a limited number of medium-size, protected segments of the molecule and condensing them to generate the full-length protein. This approach, generally referred to as a segment condensation approach (), has gained significant attention in recent years. The molecular size of the proteins that biochemists desire to design and assemble are larger compared to RNase A. Accordingly, interest in developing newer and simpler approaches of segment condensation has increased.

From genes to proteins: in vitro expression of rickettsial proteins.

The availability of the complete genome sequences of several organisms allows the comparative analysis of genomes, a branch of bioinformatics known as genomics. With this approach, much can be learned about the biology of organisms that are difficult to culture, even when few, if any, of their proteins have been isolated and studied directly. We have focused our interest on Rickettsia conorii, an obligate intracellular bacterium responsible for Mediterranean spotted fever, a disease endemic in southern Europe. While bioinformatic annotation of the complete genome of this bacteria has allowed identification of 1,374 genes, a large number of them remain functionally uncharacterized. The final goal of many experiments in molecular biology is to use biological systems to synthesize the protein encoded by the gene being studied. Because three-dimensional structures are more resilient to evolution and change than amino acid sequences, structure determination of some open reading frames should also exhibit structural similarity to previously described protein families. We have thus initiated a systematic expression and structure determination program for the proteins encoded by rickettsial genes of interest. We have cloned different genes of R. conorii by recombinational cloning (GATEWAY), Invitrogen) a method that uses in vitro site-specific recombination to accomplish a directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency. The constructions in p-Dest17 yielded several clones able to express recombinant proteins with a C-terminal histidine tag. Expression of corresponding proteins was then performed using a cell-free protein expression system (Rapid Translation System, RTS, Roche Diagnostics). The recombinational cloning approach coupled to RTS provides an approach to rapid optimization of protein expression and is very useful to express rickettsial proteins. Moreover, this system is able to overcome some of the limitations encountered with rickettsial proteins highly toxic for E. coli or insect cells.

A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells.

Whereas bacterial expression systems are widely used for production of uniformly or selectively (15)N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively (15)N-labeled proteins in insect cells. The quantities of (15)N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the (15)N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.

High-throughput cell-free protein expression systems in structural genomics/proteomics

High-throughput cell-free protein expression systems in structural genomics/proteomics

NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach

A cell-free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl–prolyl cis– trans isomerase PpiB was expressed in this system with dual amino acid-selective isotope labeling to identify the NMR signal from the active site-residue Arg87. Addition of the substrate succinyl-Ala-Ala-Pro-Phe- p-nitroanilide selectively shifted its 15N-HSQC cross peak, confirming binding to the active site. As cell-free protein expression provides high yields of protein per unit mass of labeled amino acid and sample handling is minimal, this strategy presents an exceptionally inexpensive and rapid approach to protein analysis.