Protein Synthesis Reactions Research Articles

10.2478/s11814-009-0294-4

The scale-up of cell-free protein synthesis reactions involves the preparation of large amounts of template DNA. While ion-exchange column chromatography methods have commonly been used to obtain purified plasmid DNA for cell-free protein synthesis reactions, these methods are costly and difficult to expand to a large scale. In this work, we report that the routine isopropyl alcohol (IPA) precipitation method can be used to prepare cell-free-expressible DNA when the co-precipitated proteins are removed. Compared to column-purification procedures, the IPA-precipitation offers obvious advantages with respect to the cost and scaling-up of template preparation, and we believe that our finding will contribute to making cell-free protein synthesis system more practical for the rapid production of preparative amounts of recombinant proteins.

Development of an In Vitro Compartmentalization Screen for High-Throughput Directed Evolution of [FeFe] Hydrogenases

Background[FeFe] hydrogenase enzymes catalyze the formation and dissociation of molecular hydrogen with the help of a complex prosthetic group composed of common elements. The development of energy conversion technologies based on these renewable catalysts has been hindered by their extreme oxygen sensitivity. Attempts to improve the enzymes by directed evolution have failed for want of a screening platform capable of throughputs high enough to adequately sample heavily mutated DNA libraries. In vitro compartmentalization (IVC) is a powerful method capable of screening for multiple-turnover enzymatic activity at very high throughputs. Recent advances have allowed [FeFe] hydrogenases to be expressed and activated in the cell-free protein synthesis reactions on which IVC is based; however, IVC is a demanding technique with which many enzymes have proven incompatible.Methodology/Principal FindingsHere we describe an extremely high-throughput IVC screen for oxygen-tolerant [FeFe] hydrogenases. We demonstrate that the [FeFe] hydrogenase CpI can be expressed and activated within emulsion droplets, and identify a fluorogenic substrate that links activity after oxygen exposure to the generation of a fluorescent signal. We present a screening protocol in which attachment of mutant genes and the proteins they encode to the surfaces of microbeads is followed by three separate emulsion steps for amplification, expression, and evaluation of hydrogenase mutants. We show that beads displaying active hydrogenase can be isolated by fluorescence-activated cell-sorting, and we use the method to enrich such beads from a mock library.Conclusions/Significance[FeFe] hydrogenases are the most complex enzymes to be produced by cell-free protein synthesis, and the most challenging targets to which IVC has yet been applied. The technique described here is an enabling step towards the development of biocatalysts for a biological hydrogen economy.

Comparing the functional properties of the Hsp70 chaperones, DnaK and BiP

The Hsp70 family of molecular chaperones is an essential class of chaperones that is present in many different cell types and cellular compartments. We have compared the bioactivities of the prokaryotic cytosolic Hsp70, DnaK, to that of the eukaryotic Hsp70, BiP, located in the endoplasmic reticulum (ER). Both chaperones helped to prevent protein aggregation. However, only DnaK provided enhanced refolding of denatured proteins. We also tested chaperone folding assistance during translation in the context of cell-free protein synthesis reactions for several protein targets and show that both DnaK and BiP can provide folding assistance under these conditions. Our results support previous reports suggesting that DnaK provides both post-translational and co-translational folding assistance while BiP predominately provides folding assistance that is contemporaneous with translation.

Threading the Needle: Getting Selenocysteine Into Proteins

The co-translational incorporation of selenocysteine (Sec) requires that UGA be recognized as a sense rather than a nonsense codon. This is accomplished by the concerted action of a Sec insertion sequence (SECIS) element, SECIS binding protein 2, and a ternary complex of the Sec specific elongation factor, Sec-tRNA(Sec), and GTP. The mechanism by which they alter the canonical protein synthesis reaction has been elusive. Here we present an overview of the mechanistic perspective on Sec incorporation, highlighting recent advances in the field.

Cellular Compartment Model for Exploring the Effect of the Lipidic Membrane on the Kinetics of Encapsulated Biochemical Reactions

One of the important characteristics of the cellular system is that interactions between the plasma membrane and water-soluble molecules in the cytoplasm are enhanced by the confinement of the molecules to the small volume of the intracellular space. Studying this effect in a model cell system, we measured the time evolution of an enzymatic hydrolysis reaction and a cell-free protein synthesis reaction taking place in giant liposomes having various size and phospholipid compositions by a flow cytometry. This single vesicle-based assay of a large number of liposomes enabled us to examine the volume dependence of enclosed reactions in detail, revealing that the presence of specific lipid affected the specific kinetic parameters of encapsulated reactions.

A simple and cost-effective method to prepare template DNAs for cell-free protein synthesis

The scale-up of cell-free protein synthesis reactions involves the preparation of large amounts of template DNA. While ion-exchange column chromatography methods have commonly been used to obtain purified plasmid DNA for cell-free protein synthesis reactions, these methods are costly and difficult to expand to a large scale. In this work, we report that the routine isopropyl alcohol (IPA) precipitation method can be used to prepare cell-free-expressible DNA when the co-precipitated proteins are removed. Compared to column-purification procedures, the IPA-precipitation offers obvious advantages with respect to the cost and scaling-up of template preparation, and we believe that our finding will contribute to making cell-free protein synthesis system more practical for the rapid production of preparative amounts of recombinant proteins.

Exceptional structured noncoding RNAs revealed by bacterial metagenome analysis

Estimates of the total number of bacterial species1-3 suggest that existing DNA sequence databases carry only a tiny fraction of the total amount of DNA sequence space represented by this division of life. Indeed, environmental DNA samples have been shown to encode many previously unknown classes of proteins4 and RNAs5. Bioinformatics searches6-10 of genomic DNA from bacteria commonly identify novel noncoding RNAs (ncRNAs)10-12 such as riboswitches13,14. In rare instances, RNAs that exhibit more extensive sequence and structural conservation across a wide range of bacteria are encountered15,16. Given that large structured RNAs are known to carry out complex biochemical functions such as protein synthesis and RNA processing reactions, identifying more RNAs of great size and intricate structure is likely to reveal additional biochemical functions that can be achieved by RNA. We applied an updated computational pipeline17 to discover ncRNAs that rival the known large ribozymes in size and structural complexity or that are among the most abundant RNAs in bacteria that encode them. These RNAs would have been difficult or impossible to detect without examining environmental DNA sequences, suggesting that numerous RNAs with extraordinary size, structural complexity, or other exceptional characteristics remain to be discovered in unexplored sequence space.

Cationic phosphorodiamidate morpholino oligomers efficiently prevent growth of Escherichia coli in vitro and in vivo

Phosphorodiamidate morpholino oligomers (PMOs) are uncharged DNA analogues that can inhibit bacterial growth by a gene-specific, antisense mechanism. Attaching cationic peptides to PMOs enables efficient penetration through the Gram-negative outer membrane. We hypothesized that cationic groups attached directly to the PMO would obviate the need to attach peptides. PMOs with identical 11-base sequence (AcpP) targeted to acpP (an essential gene) of Escherichia coli were synthesized with various numbers of either piperazine (Pip) or N-(6-guanidinohexanoyl)piperazine (Gux) coupled to the phosphorodiamidate linker. Peptide-PMO conjugates were made using the membrane-penetrating peptide (RXR)(4)XB (X is 6-aminohexanoic acid; B is beta-alanine). MICs (microM/mg/L) were measured using E. coli: 3 + Pip-AcpP, 160/653; 6 + Pip-AcpP, 160/673; 2 + Gux-AcpP, 20/88; 5 + Gux-AcpP, 10/49; 8 + Gux-AcpP, 10/56; 3 + Pip-AcpP-(RXR)(4)XB, 0.3/2; and 5 + Gux-AcpP-(RXR)(4)XB, 0.6/4. In cell-free protein synthesis reactions, all PMOs inhibited gene expression approximately the same. These results suggested that Pip-PMOs inefficiently penetrated the outer membrane. Indeed, the MICs of 3 + Pip-AcpP and 6 + Pip-AcpP were reduced to 0.6 and 2.5 microM (1.2 and 10.5 mg/L), respectively, using as indicator a strain with a 'leaky' outer membrane. In vivo, mice were infected intraperitoneally with E. coli. Intraperitoneal treatment with 50 mg/kg 3 + Pip-AcpP, 15 mg/kg 5 + Gux-AcpP or 0.5 mg/kg 3 + Pip-AcpP-(RXR)(4)XB, or subcutaneous treatment with 15 mg/kg 5 + Gux-AcpP or (RXR)(4)XB-AcpP reduced bacteria in blood and increased survival. Cationic PMOs inhibited bacterial growth in vitro and in vivo, and Gux-PMOs were more effective than Pip-PMOs. However, neither was as effective as the equivalent PMO-peptide conjugates. Subcutaneous treatment showed that 5 + Gux-AcpP or (RXR)(4)XB-AcpP entered the circulatory system, reduced infection and increased survival.

A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 microM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B(1-320)) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26 degrees C.

Effects of organic and inorganic zinc sources on production, composition and zinc concentration of milk in early lactating Holstein cows

Zinc (Zn) is an essential nutrient for animals, functioning largely or entirely in enzyme systems and being involved in protein synthesis, carbohydrate metabolism, and many other biochemical reactions (Miller, 1970). This element is important in maintenance of health and integrity of epithelial tissue, such as skin (teats) and mammary tissue, because of its role in cell division and protein synthesis (Cook-Mills and Fraker, 1993). The most widely used products for zinc supplementation are zinc oxide, zinc sulphate and zinc methionine. Knowledge of organic Zn compounds is still incomplete. In particular, there is a lack of reports on the effect of such compounds in roughage based diets and only a limited number of studies have investigated the importance of Zn methionine in dairy cattle. Some research suggest that supplementing certain organic forms of zinc improve animal-production responses compared with those observed in ruminants that are supplemented with inorganic zinc (Spears, 1996). Therefore, the present study was conducted to compare organic and inorganic zinc supplements effects on production and components of milk. A second objective was to determine effects of different zinc sources on zinc concentration of milk.

Quantitative Study of the Structure of Multilamellar Giant Liposomes As a Container of Protein Synthesis Reaction

Liposomes are widely used as cell-sized compartments for encapsulation of biochemical reaction systems to construct model cell systems. However, liposomes are usually diverse in both size and structure, resulting in highly heterogeneous properties as microreactors. Here, we report the development of a strategy to investigate the internal structure of giant multilamellar vesicles (GMLVs) formed by the freeze-dried empty liposomes (FDEL) method as containers of an in vitro transcription/translation system. To evaluate the occurrence of the protein synthesis reaction in GMLVs, we designed a cascade reaction system in which a synthesized enzyme hydrolyzes the fluorescent substrate, and thus the space where the reaction takes place in liposomes becomes fluorescent. We found that only a part of the liposome was reactable and not the entire internal volume, i.e., the hydrolysis reaction took place in only a part of the fractured compartment volumes in GMLVs. Simultaneous measurement of the whole internal volume of the liposomes and the quantity of reaction product of more than 100 000 liposomes using a fluorescence-activated cell sorter (FACS) revealed that the distribution of reactable volume was proportional to the whole internal volume regardless of the liposome size, i.e., the relation between the quantity of whole and reactable volume in GMLV was found to be scale-free. This information would allow us to reduce the geometric parameters of GMLV for quantitative analysis of reaction kinetics in liposomes. The present measurement and analysis method will be an indispensable tool for exploring high-dimensional properties of a model cell system based on giant liposomes.

Targeting the methionine cycle for melanoma therapy with 3‐O‐(3,4,5‐trimethoxybenzoyl)‐(−)‐epicatechin

The higher expression of methionine cycle genes in melanoma cells than in normal melanocytes may be related with increased protein synthesis and transmethylation reactions and the subsequent need for high levels of methionine. 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG), a trimethoxy derivative of epicatechin-3-gallate (ECG), effectively suppressed proliferation of melanoma cells in cultures by inducing apoptosis. TMECG modulates the expression of genes involved in methionine metabolism, cellular methylation and glutathione synthesis in melanoma cells. TMECG treatment of melanoma cells resulted in the downregulation of antiapoptotic Bcl-2, the upregulation of proapoptotic Bax and the activation of caspase-3; however, it did not induce the expression of the apoptosis protease-activating factor-1 (Apaf-1). Having elucidated the effects of TMECG on the melanoma methionine cycle, we designed therapeuthical strategies to increase its effectiveness. Combinations of TMECG with S-adenosylmethionine or compounds that modulate the intracellular concentration of adenosine strongly increase the antiproliferative effects of TMECG. The ability of TMECG to target multiple aspects related with melanoma survival, with a high degree of potency, points to its clinical value in melanoma therapy.

An Economical Method for Cell-Free Protein Synthesis using Glucose and Nucleoside Monophosphates

Cell-free protein synthesis reactions have not been seriously considered as a viable method for commercial protein production mainly because of high reagent costs and a lack of scalable technologies. Here we address the first issue by presenting a cell-free protein synthesis system with comparable protein yields that removes the most expensive substrates and lowers the cell-free reagent cost by over 75% (excluding extract, polymerase, and plasmid) while maintaining high energy levels. This system uses glucose as the energy source and nucleoside monophosphates (NMPs) in place of nucleoside triphosphates (NTPs) as the nucleotide source. High levels of nucleoside triphosphates are generated from the monophosphates within 20 min, and the subsequent energy charge is similar in reactions beginning with either NTPs or NMPs. Furthermore, significant levels (>0.2 mM) of all NTPs are still available at the end of a 3-h incubation, and the total nucleotide pool is stable throughout the reaction. The glucose/NMP reaction was scaled up to milliliter scale using a thin film approach. Significant yields of active protein were observed for two proteins of vastly different size: chloramphenicol acetyl transferase (CAT, 25 kDa) and beta-galactosidase (472 kDa). The glucose/NMP cell-free reaction system dramatically reduces reagent costs while supplying high protein yields.

Rapid Expression and Purification of 100 nmol Quantities of Active Protein Using Cell-Free Protein Synthesis

Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 +/- 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction.

Zinc in Broiler Feeding and Nutrition

Zinc is an essential trace mineral for birds, functioning elaborately in enzyme systems and being involved in protein synthesis, carbohydrate metabolism, and many other biochemical reactions. Zinc is required for normal growth, reproduction, and glandular development of birds. A severe zinc deficiency causes numerous physical and pathological changes including skin lesions, decreased growth, general disability of bones and joints, very poor feathering, reproductive failure, and reduced immunity to infection of several diseases. In skin, it is five to six times more concentrated in the epidermis than the dermis. In addition, zinc is associated with wound healing because of its role in collagen and keratin syntheses. All proliferating cells, including inflammatory cells, epithelial cells, and fibroblast, require zinc. Furthermore, zinc is an essential element of more than 200 metalloenzymes and affects their conformity, stability, and activity. The superoxide dismutase, one of the zinc-containing antioxidant enzyme, has a critical role in keeping broiler skin healthy and increasing the shelf-life of broiler meat. However, a clear appreciation of the role of this element in broiler production is still limited. This article provides an overview on the role of zinc in broiler feeding and nutrition, immunity, reproduction, and meat quality in particular.

Methylenetetrahydrofolate Reductase (MTHFR): A Novel Target for Cancer Therapy

Tumor cells have an enhanced requirement for glucose, amino acids and DNA precursors. Since folates are required for the synthesis of thymidine and purines, the metabolism of folate has been exploited as an anti-cancer target for over 6 decades, with emphasis on the inhibition of DNA synthesis. However, folate is also used to generate methionine, which is essential for proliferation by virtue of its role in protein synthesis, polyamine synthesis and transmethylation reactions. Tumor-derived cell lines and human tumor xenografts have been shown to be methionine dependent i.e., they are unable to survive without methionine and are unable to efficiently utilize homocysteine, the immediate metabolic precursor of methionine. Since non-transformed cells are methionine-independent, the targeting of methionine metabolism presents an opportunity to selectively disrupt the unique metabolic networks in cancer cells. This chapter provides an overview of the critical role of folate and methionine metabolism in tumor cells and summarizes the current anti-folate and anti-methionine strategies to inhibit growth of transformed lines and tumors. We also present our work on the development of a novel anti-cancer target, methylenetetrahydrofolate reductase (MTHFR), a key enzyme of both folate and methionine metabolism. Our data demonstrate that antisense-mediated inhibition of MTHFR is associated with increased cytotoxicity in vitro and with decreased growth of tumors in vivo. These findings warrant further investigation of this enzyme and the methionine biosynthetic pathway in exploring new strategies for cancer chemotherapy.

An In Vitro strategy for the rapid expression of recombinant proteins at low temperatures

In this study, we propose a strategy for recombinant protein expression under conditions of low-temperatures. The low-temperature production of recombinant proteins was conducted in a cell-free protein synthesis reaction through the fusion of the N-terminus of the protein of interest with that from a temperature-insensitive protein. For instance, while the expression of hlL-2 or TNF-α was negligible when the reaction temperature was below 20°C, fusion constructs of the N-terminus of these proteins with the N-terminus of CAT allowed for a substantial expression of the fused protein. This method is predicted to provide a useful approach for the expression of unstable and/or aggregation-prone protein.

Preparation of template plasmids for cell-free protein synthesis using a regenerated anion-exchange column

In this report, we describe how reactions of cell-free protein synthesis can be successfully conducted using plasmids prepared with regenerated anion-exchange columns. When washed, stripped, and equilibrated with appropriated buffers, regenerated columns were able to be used repeatedly to prepare plasmids with consistent yield and purity. The regenerated columns exhibited comparable performance to a fresh column with respect to the efficiency of protein synthesis using the plasmids prepared from them. Overall, we expect that the presented results will contribute significantly to economizing the technology of cell-free protein synthesis as a practical method for protein production in preparative scales.

An intact ribose moiety at A2602 of 23S rRNA is key to trigger peptidyl-tRNA hydrolysis during translation termination

Peptide bond formation and peptidyl-tRNA hydrolysis are the two elementary chemical reactions of protein synthesis catalyzed by the ribosomal peptidyl transferase ribozyme. Due to the combined effort of structural and biochemical studies, details of the peptidyl transfer reaction have become increasingly clearer. However, significantly less is known about the molecular events that lead to peptidyl-tRNA hydrolysis at the termination phase of translation. Here we have applied a recently introduced experimental system, which allows the ribosomal peptidyl transferase center (PTC) to be chemically engineered by the introduction of non-natural nucleoside analogs. By this approach single functional group modifications are incorporated, thus allowing their functional contributions in the PTC to be unravelled with improved precision. We show that an intact ribose sugar at the 23S rRNA residue A2602 is crucial for efficient peptidyl-tRNA hydrolysis, while having no apparent functional relevance for transpeptidation. Despite the fact that all investigated active site residues are universally conserved, the removal of the complete nucleobase or the ribose 2′-hydroxyl at A2602, U2585, U2506, A2451 or C2063 has no or only marginal inhibitory effects on the overall rate of peptidyl-tRNA hydrolysis. These findings underscore the exceptional functional importance of the ribose moiety at A2602 for triggering peptide release.

Prolonged cell‐free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate

The accumulation of inorganic phosphate inhibits protein synthesis in cell-free protein synthesis reactions that are energized by high-energy-phosphate-containing compounds. This study developed a new scheme for supplying energy using dual energy sources to enhance the regeneration of ATP and lower the rate of phosphate accumulation. In the proposed scheme, where creatine phosphate (CP) and glucose were simultaneously used as the energy sources, the phosphate released from the CP was subsequently used in the glycolytic pathway for the utilization of the glucose, which enhanced the ATP supply and reduced the rate of inorganic phosphate accumulation. When tested against different proteins, the developed method produced 2-3 times more protein than the conventional ATP regeneration methods using single energy sources.