Wheat Germ Cell-free Protein-synthesizing System Research Articles

Application of a Cell-Free Synthetic Biology Platform for the Reconstitution of Teleocidin B and UK-2A Precursor Biosynthetic Pathways.

We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.

Leveraging the wheat germ cell-free protein synthesis system to accelerate malaria vaccine development.

Vaccines against infectious diseases have had great successes in the history of public health. Major breakthroughs have occurred in the development of vaccine-based interventions against viral and bacterial pathogens through the application of classical vaccine design strategies. In contrast the development of a malaria vaccine has been slow. Plasmodium falciparum malaria affects millions of people with nearly half of the world population at risk of infection. Decades of dedicated research has taught us that developing an effective vaccine will be time consuming, challenging, and expensive. Nevertheless, recent advancements such as the optimization of robust protein synthesis platforms, high-throughput immunoscreening approaches, reverse vaccinology, structural design of immunogens, lymphocyte repertoire sequencing, and the utilization of artificial intelligence, have renewed the prospects of an accelerated discovery of the key antigens in malaria. A deeper understanding of the major factors underlying the immunological and molecular mechanisms of malaria might provide a comprehensive approach to identifying novel and highly efficacious vaccines. In this review we discuss progress in novel antigen discoveries that leverage on the wheat germ cell-free protein synthesis system (WGCFS) to accelerate malaria vaccine development.

Cloning and in vitro and in vivo expression of plant glutathione S-transferase zeta class genes

The cDNAs encoding the zeta class of glutathione S-transferases (GSTs) ( GSTZs), which are multifunctional enzymes, were cloned from Arabidopsis thaliana L. and three lines of Brassica napus L. by RT PCR, and named AtGSTZ for A. thaliana ecotype Columbia gll, and BnGSTZ-A, BnGSTZ-B and BnGSTZ-C for B. napus cv. Shan 2A, Shan 213 and Ken C1, respectively. Sequence analysis revealed that the sequences of Shan 2A and Shan 2B were identical, and Ken C1 was different only in 3.8% of the sequence. Comparison of these sequences with published sequences in GeneBank showed that the sequences of the Brassica species were unpublished. The cDNAs were then inserted into two vectors for in vivo expression in Escherichia coli and in vitro expression in a wheat-germ cell-free protein synthesis system. All GSTZs were well expressed both in vivo and in vitro as an enzymatically active form, showing that all of them had both dichloroacetic acid dechlorinating and maleylacetone isomerase activities.

Cloning and in Vitro and in Vivo Expression of Plant Glutathione S-Transferase Zeta Class Genes

The cDNAs encoding the zeta class of glutathione S-transferases (GSTs) (GSTZs), which are multifunctional enzymes, were cloned from Arabidopsis thaliana L. and three lines of Brassica napus L. by RT PCR, and named AtGSTZ for A. thaliana ecotype Columbia gll, and BnGSTZ-A, BnGSTZ-B and BnGSTZ-C for B. napus cv. Shan 2A, Shan 213 and Ken C1, respectively. Sequence analysis revealed that the sequences of Shan 2A and Shan 2B were identical, and Ken C1 was different only in 3.8% of the sequence. Comparison of these sequences with published sequences in GeneBank showed that the sequences of the Brassica species were unpublished. The cDNAs were then inserted into two vectors for in vivo expression in Escherichia coli and in vitro expression in a wheat-germ cell-free protein synthesis system. All GSTZs were well expressed both in vivo and in vitro as an enzymatically active form, showing that all of them had both dichloroacetic acid dechlorinating and maleylacetone isomerase activities.

Can ACG serve as an initiation codon for protein synthesis in eucaryotic cells?

An ACG codon, which replaces the AUG codon used to initiate the synthesis of bacteriophage T7 gene 0.3 protein, was shown to function as a low-efficiency initiation codon in a wheat germ cell-free protein-synthesizing system.

Can ACG serve as an initiation codon for protein synthesis in eucaryotic cells?

An ACG codon, which replaces the AUG codon used to initiate the synthesis of bacteriophage T7 gene 0.3 protein, was shown to function as a low-efficiency initiation codon in a wheat germ cell-free protein-synthesizing system.

Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells

Acute hormonal effects on the synthesis rate of the cytosolic form of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP), were investigated using rat hepatocytes maintained in short-term suspension culture. Cells were pulse-labeled with [3H]leucine or [35S]methionine and the rate of synthesis of phosphoenolpyruvate carboxykinase was estimated after immunoprecipitation of cell extracts with specific antibodies or following high-resolution two-dimensional gel electrophoresis of cell proteins. Total RNA was also extracted from cultured cells and subsequently translated in a wheat germ cell-free protein-synthesis system, in order to quantify the level of functional mRNA coding for phosphoenolpyruvate carboxykinase. Glucagon, the single most effective inducer, causes a 15--20-fold increase in the level of specific mRNA in 2 h, accompanied by a similar increase in enzyme synthesis rate. The extent of induction is further amplified about threefold when dexamethasone is added to the culture medium. The synergistic action of dexamethasone does not require pre-exposure of the cells to the glucocorticoid, but on the contrary occurs without lag upon simultaneous addition of glucagon and dexamethasone. The induction of phosphoenolpyruvate carboxykinase mRNA by glucagon is markedly depressed in hepatocytes inhibited for protein synthesis by cycloheximide. Cycloheximide-inhibited cells, however, display a considerable induction of the message after joint stimulation with dexamethasone and glucagon. Thus, the synergistic action of dexamethasone does not require concomitant protein synthesis. These data provide indirect evidence for a primary effect of the glucocorticoids on the expression of the phosphoenolpyruvate carboxykinase gene. Besides glucagon and dexamethasone, the thyroid hormones are shown to influence the rate of phosphoenolpyruvate carboxykinase synthesis in isolated liver cells. The stimulatory effect of 3,5,3'-triiodothyronine (T3) is best demonstrated as a twofold increase in relative rate of enzyme synthesis in cells supplied with T3 plus glucagon, as compared to cells challenged with glucagon alone. The effect of T3 relies on a pretranslational mechanism, as shown by a commensurate increase in functional mRNA coding for phosphoenolpyruvate carboxykinase. Dose-response experiments with T3 as well as dexamethasone demonstrate effects at very low hormone levels, consistent with a role for these hormones as physiological modulators of phosphoenolpyruvate carboxykinase expression.

Cell-free translation of polyribosomes from detached pumpkin cotyledons: Effects of starvation and cytokinin

The effects of 6-benzylaminopurine (BAP, 5.10−5M) treatment of pumpkin cotyledons and their starvation after excision upon polysome/monosome ratio and translational capacity of polysomes in cell-free system were studied. It has been found that starvation causes a progressive polysome degradation. Polysome translation in a wheat germ cell-free proteinsynthesizing system reveals that the translation capacity of polysome preparations decreases with the time after cotyledon excision much more sharply than polysome/monosome ratio. This indicates the starvation damage in elongation steps of protein synthesis. The decrease of postribosomal supernatants activity in the system of poly(U)-directed polyphenylalanine synthesis confirms this conclusion.

Cell-Free Synthesis of Wheat Prolamins

Total RNA, polyribosomes and polyribosomal RNA have been isolated from immature wheat grain and used to prime the wheat-germ cell-free protein synthesis system to produce storage protein. The synthesis of wheat prolamins, a major fraction of storage proteins, was mainly associated with membrane-bound polyribosomes. The ratio of bound to free polyribosomes increased from one at 8 days to approximately three at 32 days post-anthesis. SDS polyacrylamide gel electrophoresis and fluorography of the prolamins showed that the same components were being synthesized and accumulated from 12 days post-anthesis onward. Evidence is presented that prolamins within the molecular weight range 35 000-45 000 may be translated with a signal sequence attached. These signal sequences are rapidly removed during translation and further post-translational modifications may occur over longer periods.

14] Isolation and translation of mouse interferon mRNAs

It has been observed that the wheat germ cell-free protein-synthesizing system translates mouse interferon mRNA to protein that was active in inhibiting viral multiplication in mouse cells. Easy availability of the wheat embryo and the simplicity of the procedure for the preparation of the extract make this material a good choice for studies of protein synthesis. Mouse interferon mRNAs isolated by the two methods as discussed in this chapter are translated in the wheat germ extract with high efficiency. The fidelity of the translation is reflected in the production of the biologically active product. A high efficiency of translation of mouse interferon mRNA to biologically active protein in the wheat germ ceil-free system needs the presence of polyamines. Translation of many plant viral RNAs in the wheat germ extract is stimulated by addition of polyamines such as spermine or spermidine. It is important to maximize the translation system for both the ionic requirements and the polyamine concentration. The product of translation of mouse mRNA in the wheat germ extract is characterized as mouse interferon by (a) its inhibition of virus multiplication in homologous cells, (b) its absence of activity in heterologous cells, (c) its neutralization by anti-mouse interferon antiserum, and (d) the absence of inhibitory effects on viral multiplication by the translation products obtained from mRNA isolated from non-induced cells.

In vitro biosynthesis of somatostatin. Evidence for two distinct preprosomatostatin molecules.

mRNA isolated from angler fish islets of Langerhans was translated in the wheat germ cell-free protein-synthesizing system and the products identified by immunoprecipitation with specific antibodies to somatostatin followed by sodium dodecyl sulfate gel electrophoresis. As previously shown (Shields, d. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 4074), a major polypeptide of 18,000 dalton, designated preprosomatostatin, was immunoprecipitable. Here, evidence is presented for an additional somatostatin-immunoreactive polypeptide of apparent Mr = 19,000. The 19 kilodalton polypeptide was similar, but not identical with the 18 kilodalton preprosomatostatin, as determined by tryptic peptide analysis. Comparison of the tryptic peptides of the 19,000 dalton polypeptide with those of unlabeled somatostatin demonstrated that it contained the authentic somatostatin sequence. Like the 18,000 dalton precursor, the 19,000 dalton polypeptide had the mature somatostatin sequence located at its COOH terminus; it is proposed that this molecule is a minor species of preprosomatostatin.

Antagonistic action between antibodies directed against 7-methylguanosine and polyamines on translation in vitro of RNA induced by measles virus.

Antibodies directed against N7-methylguanosine (m7Guo) were prepared and added to a wheat germ cell-free protein-synthesizing system programmed with RNA extracted from monkey cells persistently infected with measles virus. A dose-dependent inhibition of [35S]methionine incorporation was observed when RNA was preincubated with anti-m7Guo immunoglobulins. Antibodies preincubated with m7Guo did not show any inhibiting activity. The inhibitory effect of antibodies was abolished when RNA was preincubated with immunoglobulins in the presence of spermine and spermidine. When polyamines were added to the assay programmed with the IgG-RNA complex, no inhibition was observed.

Regulation of protein synthesis during spore germination in Dictyostelium discoideum.

Spore germination in the slime mold Dictyostelium discoideum is a particularly suitable paradigm for studying the regulation of gene expression because developmentally regulated changes in both protein and mRNA synthesis occur during the synchronous transition from dormant spore to growing ameba. To investigate the regulation of protein synthesis during germination, we labeled activated spores with [35S]methionine at 1-hr intervals during germination, until amebas emerged (at 3 hr). The labeled proteins were resolved by two-dimensional polyacrylamide gel electrophoresis. Six classes of proteins were distinguished, depending on the time of onset and duration of their synthesis: (i) proteins made only during the first hour of germination, (ii) proteins made during the second hour, (iii) proteins made during the third hour, (iv) those synthesized only between 1 and 3 hr after activation, (v) peptides made only between 0 and 2 hr after activation, and (vi) proteins that were made throughout germination, mRNA isolated from dormant spores and from spores at different stages of germination was translated in a wheat germ cell-free protein-synthesizing system, and the proteins made in vitro were compared to those synthesized in vivo. The majority of the changes in the pattern of protein synthesis that occurred during the different stages of germination were attributable to the presence or absence of translatable mRNA. It is concluded that the synthesis of a majority of the proteins during spore germination is transcriptionally controlled.

Initiation and processing in vitro of the primary translation products of guinea-pig caseins

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal 'signal'-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

Translation and characterization of messenger RNAs in differentiating chicken cartilage.

Total RNA, prepared from chicken limb bud cultures undergoing differentiation to cartilage, has been translated in a wheat germ cell-free protein-synthesizing system. Antibodies against chondroitin sulfate proteoglycan core protein immunoprecipitate a single component which migrates as a protein of 340,000 daltons in sodium dodecyl sulfate/polyacrylamide gels. The messenger RNA for this protein sediments at approximately 27 S in 70% formamide or aqueous sucrose gradients. The 340,000-dalton protein is present in cell-free translation products directed by RNA prepared from limb bud cultures and sternae and is absent in cell-free translation directed by RNA prepared from embryonic calvaria or liver. The level of synthesis of this protein is greatly reduced when RNA prepared from limb bud cultures inhibited from differentiation by BrdUrd is used. (Pre)pro alpha 1(I), -alpha 2(I), and -alpha 1(II) collagen bands have been identified on gels by electrophoresis of collagenase-digested or immunoprecipitated cell-free translation products directed by RNA from differentiating limb bud cultures, embryonic sternae, and embryonic calvaria.

Isolation and translation in vitro of poly(A)+ RNA from the free-living nematode Panagrellus silusiae

Polysomal RNA was isolated from the free-living nematode Panagrellus silusiae. Passage of this RNA through a cellulose column resulted in the fractionation of the input RNA into poly(A)-RNA (ca. 97.5% of the total) and poly(A)+ RNA (ca. 2.5% of the total). RNase digestion, followed by polyacrylamide gel electrophoresis, revealed that the poly(A)+ RNA contained poly(A) tracts that ranged from 75 to 104 nucleotides in length with a mean value of about 90 residues. There was no evidence of poly(A) sequences in the poly(A)- RNA fraction. Poly(A)+ RNA gave a 25- to 50-fold stimulation (over background) of amino acid incorporation in the wheat germ cell-free protein-synthesizing system. At least 26 proteins were evident after electrophoresis in cylindrical sodium dodecyl sulfate-polyacrylamide gels. Poly(A)-RNA was capable of stimulating protein synthesis in vitro with about five discrete proteins being produced. In summary, the properties of mRNA from a simple organism such as P. silusiae are very similar to those of more complex eukaryotes.

The effect of anti-spermine antibodies on a wheat germ cell-free protein-synthesizing system

FEBS LettersVolume 99, Issue 1 p. 20-24 Full-length articleFree Access The effect of anti-spermine antibodies on a wheat germ cell-free protein-synthesizing system A. Niveleau, A. Niveleau Unité de Virologie Fondamentale et Appliquée, INSERM (U51) Groupe de Recherche CNRS 33, 1, place Professeur Joseph Renaut, 69371 Lyon Cedex 2, FranceSearch for more papers by this authorG.A. Quash, G.A. Quash Unité de Virologie Fondamentale et Appliquée, INSERM (U51) Groupe de Recherche CNRS 33, 1, place Professeur Joseph Renaut, 69371 Lyon Cedex 2, FranceSearch for more papers by this author A. Niveleau, A. Niveleau Unité de Virologie Fondamentale et Appliquée, INSERM (U51) Groupe de Recherche CNRS 33, 1, place Professeur Joseph Renaut, 69371 Lyon Cedex 2, FranceSearch for more papers by this authorG.A. Quash, G.A. Quash Unité de Virologie Fondamentale et Appliquée, INSERM (U51) Groupe de Recherche CNRS 33, 1, place Professeur Joseph Renaut, 69371 Lyon Cedex 2, FranceSearch for more papers by this author First published: March 01, 1979 https://doi.org/10.1016/0014-5793(79)80239-2Citations: 8AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume99, Issue1March 01, 1979Pages 20-24 ReferencesRelatedInformation

Cell-free synthesis of metallothionein directed by rat liver polyadenylated messenger ribonucleic acid.

Total RNA was isolated from rat liver polyribosomes and fractionated by oligo(dT)-cellulose chromatography to obtain polyadenylated mRNA. The mRNA was translated in a wheat-germ cell-free protein-synthesizing system containing [3H]glycine, [3H]lysine and [3H]serine. Most of the newly synthesized 3H-labelled polypeptides were removed from the cell-free products by precipitation at pH 4.0. 3H-labelled thionein chains, which were soluble at pH 4.0, were purified by activated-thiol-Sepharose 4B chromatography or by gel-filtration chromatography. Polyribosomal thionein mRNA was found to increase by at least 3-fold after parenteral administration and by 20 h thereafter the ratio of thionein mRNA to total mRNA approached that found in controls. Actinomycin D administration in vivo blocked the Zn2+-induced increase in polyribosomal thionein mRNA content. These data strongly suggest that metallothionein is an inducible protein. The mechanism of regulation appears to involve changes in the synthesis de novo of thionein mRNA and hence the pool of thionein mRNA available for translation.

Isolation and characterization of two mRNAs from HeLa S 3 cells coding for histone H4

Summary Histone mRNAs from S phase HeLa S 3 cells were fractionated electrophoretically in a 6% acrylamide slab gel. We have isolated two RNA species, both of which code for H4 histone when translated in a wheat germ cell-free protein-synthesizing system. Using oligo dT cellulose chromatography we have shown that neither of the RNA species contains poly A. Electrophoretic fractionation under denaturing conditions in 98% formamide-8% acrylamide gel indicates that the two H4 histone mRNAs differ in molecular weight.

Polypeptides specified by the influenza virus genome 2. Assignment of protein coding functions to individual genome segments by in vitro translation

Cytoplasmic RNA extracted from chick embryo fibroblasts infected with influenza A (fowl plague) virus (FPV) was translated in a wheat germ cell-free protein-synthesizing system. Polypeptides which comigrated during SDS-polyacrylamide gel electrophoresis with marker virus-specific polypeptides P 1, P 2, P3, NP, M, and NS were synthesized in vitro. The NP, M, and NS polypeptides were positively identified by tryptic peptide mapping. The polypeptide component of the virus glycoprotein HA was also synthesized in vitro, and was identified by tryptic peptide mapping. RNA extracted from purified FPV (vRNA) did not direct the synthesis of any recognizable virus-specific polypeptides in vitro, either as a total preparation, or as individual RNA genome segments. The protein coding functions of the vRNA segments were identified by hybridization of individual segments to a preparation of infected cell cytoplasmic RNA. On subsequent translation of the RNA in vitro, synthesis of the virus-specific polypeptide corresponding to the hybridized vRNA segment was specifically reduced. We conclude that, for FPV, virion RNA segments 1–3 code for the three P polypeptides and segments 4, 5, 6, 7, and 8 code for polypeptides HA, NP, NA, M, and NS, respectively.