Effect of hypoxia on nucleic acid and protein synthesis in different brain regions

The incorporation of [methyl-³H]thymidine into DNA, of [5-³H]uridine into RNA, and of [1-¹⁴C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.     

A simple,reproducible cell-free system for measuring brain protein synthesis

A simple, rapid, sensitive, and reproducible cell-free assay system for studying brain protein synthesis is described. This system uses small amounts of brain postmitochondrial supernatant, making it a convenient screening test when only small amounts of tissue are available. It showed over 95% dependence on Mg²⁺ and on an energy source. Optimal incorporation occurred under the following conditions: Mg²⁺ 3 mM; ATP, 0.6 mM; GTP, 0.6 mM; high K⁺, 25 mM; Low Na⁺, 15 mM; pH 7.1–7.5. The rate of amino acid incorporation did not vary with leucine concentrations in vitro up to 1 mM, which obviated the need to measure endogenous leucine concentrations.     

Selenomethionine incorporation into a protein by cell-free synthesis

Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.     

Mitochondrial DNA,RNA, and protein synthesis in different regions of developing rat brain

In vivo and in vitro (tissue slices) incorporation of labeled precursors into DNA, RNA, and proteins was measured in mitochondria obtained from cerebral hemispheres, cerebellum, and brain stem of rats at different days of postnatal development. To compare the synthesis of macromolecules in mitochondria with that in other subcellular fractions, the incorporation of labeled precursors into DNA, RNA, and proteins extracted from nuclei and into RNA and proteins extracted from microsomes and cytoplasmic soluble fractions was also measured.The results obtained showed that the incorporation of [³H]thymidine into DNA and of [¹⁴C]leucine into proteins of nuclei and mitochondria from the various brain regions examined decreased during postnatal development, however, at 30 days of age the specific radioactivity of mitochondrial DNA was higher than that of nuclear DNA. [³H]Uridine incorporation into RNA decreased from 10 to 30 days of age in nuclei while in mitochondria it was quite similar at both ages. This result may be due to a faster turnover of mitochondrial RNA compared to that of mitochondrial DNA and proteins. The results obtained suggest an active biosynthesis of macromolecules in brain mitochondria and might indicate an intense biogenesis of these organelles in rat brain during postnatal development.Preliminary reports of these results were presented at the XI FEBS Meeting, Copenhagen, August 14–19, 1977, Poster number A2-2-155-3, and at III Meeting of Italian Biochem. Soc., Siena, October 3–5, 1977, Abstract C6.     

Prolonging cell-free protein synthesis by selective reagent additions

Factors causing the early cessation of protein synthesis have been studied in a cell-free system from Escherichia coli. We discovered that phosphoenol pyruvate (PEP), the secondary energy source for ATP regeneration, and several amino acids are rapidly degraded during the cell-free protein synthesis reaction. The degradation of such compounds takes place even in the absence of protein synthesis. This degradation severely reduces the capacity for protein synthesis. The lost potency was completely recovered when the reaction mixture was supplied with additional PEP and amino acids. Of the 20 amino acids, only arginine, cysteine, and tryptophan were required to restore system activity. Through repeated additions of PEP, arginine, cysteine,and tryptophan, the duration of protein synthesis was greatly extended. In this fed-batch reaction, after a 2 h incubation, the level of cell-free synthesized chloramphenicol acetyl transferase (CAT) reached 350 microg/mL, which is 3.5 times the yield of the batch reaction. Addition of fresh magnesium further extended the protein synthesis. As a result, through coordinated additions of PEP, arginine, cysteine, tryptophan, and magnesium, the final concentration of cell-free synthesized CAT increased more than 4-fold compared to a batch reaction. SDS-PAGE analysis of such a fed-batch reaction produced an obvious band of CAT upon Coomassie Blue staining.     

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.     

Translation of chicken interferon messenger in a cell-free protein synthesis system and in a cell culture

A highly effective cell-free system for protein synthesis was obtained from rabbit reticulocytes and for the first time used for synthesis of biologically active chicken interferon. The optimal conditions for translation of its mRNA were developed. The translation efficacy in the cell-free system was 10-50 times higher than that in the culture of heterologous cells. The higher the purity level of RNA, the higher the translation level. With respect to poly (A+) RNA sedimenting in the sucrose gradient 9S the efficacy reached 2560 units per 1 microgram of RNA. By the content of poly (A), sequences and rate of the sedimentation, mRNA of the chicken interferon was similar to that of the human fibroblast cell interferon. The possible translation of mRNA of the chicken interferon at low concentrations of exogenic potassium ions in the cell-free system is explained by production of interferon in infected cells where the concentration of the intracellular potassium significantly decreases which is indicative of the mRNA interferon similarity with virus templates. It was found that only albino New Zealand rabbits, but also chinchilla may be used for preparation of the cell-free protein synthesizing system. Various exogenic templates in the mRNA-dependent cell-free system prepared from reticulocyte nonfractionated lysate by treatment with micrococcal nuclease stimulated the protein synthesis by 7-15 times.     

Inhibition of eukaryotic cell-free protein synthesis by thionins from wheat endosperm

Thionins are polypeptide toxins of about 5000 molecular weight, present in the endosperms of many Gramineae, which modify membrane permeability and inhibit macromolecular synthesis in cultured mammalian cells. Evidence is presented that they inhibit in vitro protein synthesis at micromolar concentrations in cell-free systems derived from wheat germ or from rabbit reticulocytes. Inhibition seems to occur by direct binding of mRNA by the toxin, as judged by the ability of thionins to mediate retention of RNA in nitrocellulose filters and by the dependence of inhibitory concentrations on the amount of exogenous RNA added to the wheat-germ translation system. Commercial preparations of wheat-germ have been found to include some endosperm contamination (up to 15%), which may result in at least partially inhibitory concentrations of the toxin in the cell-free extracts.     

Macromolecular synthesis in mitochondria isolated from different regions of developing rat brain

DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-old rats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.     

Streamlined cell-free protein synthesis from sequence information

In this study, we describe a cell-free protein synthesis consolidated with polymerase chain reaction (PCR)-based synthetic gene assembly that allows for streamlined translation of genetic information. In silico-designed fragments of target genes were PCR-assembled and directly expressed in a cell-free synthesis system to generate functional proteins. This method bypasses the procedures required in conventional cell-based gene expression methods, integrates gene synthesis and cell-free protein synthesis, shortens the time to protein production, and allows for facile regulation of gene expression by manipulating the oligomer sequences used for gene synthesis. The strategy proposed herein expands the flexibility and throughput of the protein synthesis process, a fundamental component in the construction of synthetic biological systems.     

Improving cell-free protein synthesis for stable-isotope labeling

Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium l-glutamate (l-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium d-glutamate (d-Glu system). The productivity of the d-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the ¹H–¹⁵N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the d-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the d-Glu system. These results indicate that the d-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

无细胞体系非天然蛋白质合成研究进展

无细胞非天然蛋白质合成作为蛋白质研究的新兴手段,已成功用于表征蛋白质分子间、蛋白质与核酸分子间相互作用等基础科学研究及医药蛋白、蛋白质材料等工业生产领域。无细胞非天然蛋白质合成系统不需维持细胞的生长,无细胞膜阻碍,可依据研究目的添加基因元件或化学物质从而增强工程设计和过程调控的自由性;也可赋予蛋白质新的特性、结构及功能,如可实现蛋白翻译后修饰、反应手柄引入、生物物理探针及多聚蛋白质合成等。文中系统地综述了目前应用于无细胞蛋白质合成系统中的非天然氨基酸嵌入方法,包括全局抑制及基于正交翻译体系的终止密码子抑制、移码抑制、有义密码子再分配和非天然碱基等方法的研究进展,及非天然氨基酸在蛋白质修饰、生物物理探针、酶工程、蛋白质材料以及医药蛋白质生产等领域的应用进展,并分析了该体系的发展前景及广泛工业化应用的机遇与挑战。     

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.     

Energizing cell-free protein synthesis with glucose metabolism

In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.