Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli

The utilization efficiency of a secondary energy source in a cell-free protein synthesis system can be improved by use of a metabolic inhibitor. Oxalate, a potent inhibitor of phophoenolpyruvate synthetase, substantially increased the yield of chloramphenicol acetyltransferase synthesis through the enhanced supply of ATP. Oxalate, at 2.7 mM, increased the synthesis yield by 47% when successive amino acids additions prevent amino acid depletion during protein synthesis. These results suggest that cell-free protein synthesis efficiency could also be improved by disrupting the gene encoding phosphoenolpyruvate synthetase.     

An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

In vitro translation systems are used to investigate translational mechanisms and to synthesize proteins for characterization. Most available mammalian cell-free systems have reduced efficiency due to decreased translation initiation caused by phosphorylation of the initiation factor eIF2alpha on Ser51. We describe here a novel cell-free protein synthesis system using extracts from cultured mouse embryonic fibroblasts that are homozygous for the Ser51 to- Ala mutation in eIF2alpha (A/A cells). The translation efficiency of a capped and polyadenylated firefly luciferase mRNA in A/A cell extracts was 30-fold higher than in wild-type extracts. Protein synthesis in extracts from A/A cells was active for at least 2 h and generated up to 20 microg/mL of luciferase protein. Additionally, the A/A cell-free system faithfully recapitulated the selectivity of in vivo translation for mRNA features; translation was stimulated by a 5'-end cap (m7GpppN) and a 3'-end poly(A) tail in a synergistic manner. The system also showed similar efficiencies of cap-dependent and IRES-mediated translation (EMCV IRES). Significantly, the A/A cell-free system supported the post-translational modification of proteins, as shown by glycosylation of the HIV type-1 gp120 and cleavage of the signal peptide from beta-lactamase. We propose that cell-free systems from A/A cells can be a useful tool for investigating mechanisms of mammalian mRNA translation and for the production of recombinant proteins for molecular studies. In addition, cell-free systems from differentiated cells with the Ser51Ala mutation should provide a means for investigating cell type-specific features of protein synthesis.     

植物磷稳态的调控机制

磷是植物必需的重要营养元素之一,是生物大分子的重要组成部分,在植物生命过程中发挥着不可或缺的作用.维持体内磷稳态对于植物的生长发育和环境应答至关重要.多种信号分子参与调控植物对磷的吸收和转运.植物维持磷稳态主要包括土壤磷的活化、磷的吸收、转运、存储和再利用等过程,涉及磷胁迫响应、转录因子调节、miRNA调节、菌根共生、...     

无细胞蛋白质合成系统

无细胞蛋白质合成系统相比传统的细胞表达系统有许多优点,包括表达周期短、反应条件容易控制等。该文介绍了无细胞蛋白质合成系统的技术进展及表达控制,并综述了这一系统的应用发展。     

A highly efficient and economical cell-free protein synthesis system using the S12 extract of <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have developed an economical and simple cell-free protein synthesis system that produces milligram quantities of proteins in a milliliter batch reaction. In this system, the S12 extract, which was prepared from glucose-adapted cells, was employed and glucose alone was successfully used for the efficient and stable regeneration of ATP. The ATP level in the reaction mixture remained stable over a remarkably extended reaction period, which enabled prolonged protein synthesis, and the issues associated with proton accumulation and amino acid depletion were simultaneously addressed. Under the reaction conditions established in this study, protein synthesis continued for 6 h and the amount of the accumulated protein reached 1.8 mg/mL.     

Development of a cell-free translation system from Cucumis melo: preparation, optimization and evaluation of sensitivity to some translational inhibitors

A very active cell-free translation system was prepared from 4–5-day-old embryonic axes of melon (Cucumis melo L.), a species whose dry seeds contain a powerful translational inhibitor. The system was optimized for Mg²⁺, K⁺, NH₄⁺, high speed supernatant, total wheat germ tRNA, time and temperature. Using a 30 000 × g supernatant, the system translates endogenous messengers and polyuridylic acid very efficiently. Melon ribosomes were inhibited in vitro by several well-known eukaryotic inhibitors including melonin, the protein inhibitor present in the dry seeds of C. melo. Our results suggest that the protein inhibitor does not affect the activity of melon ribosomes neither in vivo nor during their isolation.     

自组装蛋白质芯片——功能蛋白质组学的新工具

传统的蛋白质芯片制备需要进行繁琐的蛋白质表达与纯化。同时,由于蛋白质活性不稳定,蛋白质芯片不宜长期保存。新一代自组装蛋白质芯片,利用无细胞表达体系和DNA固定技术,能够将蛋白质即时、原位表达并固定在芯片上,有效地解决了传统蛋白质芯片的制备和保存问题。目前自组装蛋白质芯片已初步用于大规模蛋白-蛋白质相互作用的筛选,以及鉴定免疫优势抗原等研究。该文介绍了近年自组装蛋白质芯片技术的进展和应用研究。     

无细胞系统的基因表达与蛋白质合成

无细胞翻译——利用细胞提取液在体外合成蛋白质,已是国外分子生物学实验室的一项常规技术,但在国内此项技术的利用却几乎是空白.对体外无细胞系统的特性、功能、优缺点及其进展等进行了全面的介绍,以期使国内学者了解和利用这一方便而有效的表达系统,进行应用生物化学与分子生物学的实验研究.     

An <Emphasis Type="Italic">In Vitro</Emphasis> 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.     

Role of Hydroxyl Group at C-2 on Pyranoid Moiety in Interaction with Pea Phytohemagglutinin

A simplified procedure for the purification of pea phytohemagglutinin, which involves fractionation with ammonium sulfate and column chromatography to give two active components, has been developed. The main component of the purified hemagglutinin gave one single band with disc electrophoresis. Molecular weight of the hemagglutinin was calculated as 61,000 (Vρ = 0.75). Using this hemagglutinin, the experiment was carried out to aim from the viewpoint of conformational feature of sugars at an explanation of the role of a hydroxyl group on gluco- or mannopyranoid moiety of the sugars capable of interaction with the hemagglutinin. A possible concern of orientation of hydroxyl group at the C–2 position on pyranoid moiety of sugars is proposed to interpret the hemagglutination with the phytohemagglutinin from the results of inhibition tests and from the consideration on conformational feature of sugars reported previously.