Translation of mouse interferon messenger RNA in a cell-free system

Crude mouse interferon mRNA extracted from poly (I) . poly (C)-induced L929 cells has been translated in a cell-free system from rabbit reticulocytes and in two-cell systems--L929 cells and chick embryo fibroblasts. Translational efficiency of interferon mRNA preparation was 100-fold higher in the cell-free system than in tissue culture cells. Interferon synthesized was species-specific and its antiviral activity was completely neutralized by mouse interferon antiserum.     

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.     

Protein synthesis in chloroplasts: V. Translation of messenger RNA for the large subunit of fraction I protein in a heterologous cell-free system

The addition of spinach chloroplast total RNA to cell-free extracts from Escherichia coli stimulates amino acid incorporation into protein. The products were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and were qualitatively and quantitatively similar to those synthesized in intact isolated chloroplasts. There are two major discrete products of both systems with molecular weights of 52,000 and 35,000. The [³⁵S]methionine-containing chymotryptic peptides of the 52,000 M_r polypeptide synthesized in the E. coli cell-free system have been compared with those of fraction I protein large subunit labelled with [³⁵S]methionine in vivo. From the close similarity in chromatographic properties of the peptides of the two polypeptides, we conclude that the 52,000 M_r product of chloroplast RNA-directed protein synthesis in E. coli extracts is the large subunit of fraction I protein.     

Translation of messenger RNA of pig kidney D-amino acid oxidase in a cell-free system

In vitro synthesis of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3], one of the peroxisomal flavin enzymes, was performed using a rabbit reticulocyte lysate system in order to elucidate the biosynthetic pathway of the enzyme. The apparent molecular weight of the synthesized enzyme protein was the same as that of D-amino acid oxidase purified from pig kidney. On the other hand, the enzyme protein was not detectable when a wheat germ lysate system was used for the translation. Denaturation of pig kidney poly(A)+ RNA with methylmercury hydroxide prior to the translation was found to enhance the synthesis of the enzyme protein. These results suggest a tight conformational structure of the mRNA used.     

Translation of collagen messenger RNA in a cell-free system derived from wheat germ.

A cell-free system for synthesizing protein from wheat germ was used to translate the messenger RNA extracted from 16-day embryonic chick calvaria. A part of the product had properties similar to collagenous peptides and served as a substrate for prolyl hydroxylase, an enzyme specific for collagen. The level of potassium was critical for the synthesis of high molecular weight products with properties similar to pro-alpha-chains. The potassium concentration for optimal protein synthesis, as judged by maximum incorporation of [3H]proline into acid precipitable material, was considerably lower than the concentration required for the synthesis of high molecular weight collagenous peptides.     

Translation of albumin messenger RNA in a cell-free protein-synthesizing system derived from wheat germ.

Purified rat liver albumin mRNA directed the synthesis of albumin in a mRNA-dependent cell-free protein-synthesizing system derived from wheat germ extracts. The [3H]leucine-labeled in vitro translation product reacted with antibodies specific for albumin and co-migrated with authentic 14C-labeled serum albumin during gel electrophoresis in the presence or absence of sodium dodecyl sufate. Higher concentrations of potassium and magnesium ions were required for the translation of albumin mRNA than for total liver mRNAs. These requirements were consistent for the purified albumin as well as when it was a component in the liver mRNA mixture. At the higher potassium or magnesium concentrations, only intact albumin molecules were synthesized, whereas lower concentrations of these ions caused the production of antibody-reactive fragments. These fragments were apparently the result of premature termination of peptide synthesis and not due to endogenous proteolytic activity.     

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.     

无细胞蛋白合成体系实现胰岛素原可溶性表达

胰岛素原(Proinsulin,Pins)是胰岛素的合成前体。在大肠杆菌表达系统中,其一般以包涵体的形式存在,需要经过变性复性等后续加工过程才能得到有活性的胰岛素。而无细胞蛋白合成体系(Cell-free protein synthesis,CFPS)作为一种新型体外蛋白合成手段,突破了细胞的生理限制,已成功应用于多种重组蛋白药物的生产。为了探索胰岛素合成的新方法以满足其在新型给药途径研发中的需求,本研究运用CFPS体系进行胰岛素原的可溶性表达。通过将胰岛素原与荧光蛋白进行融合来增加其可溶性,成功在CFPS体系中表达了胰岛素原融合蛋白。最后使用Western blotting对融合红色荧光蛋白的胰岛素原(Pins-mCherry)进行鉴定,利用酶标仪对融合绿色荧光蛋白的胰岛素原(Pins-eGFP)在上清中的表达进行定量分析,结果表明Pins-eGFP部分可溶,其表达量为(12.28±3.45)μg/m L。本研究首次实现了融合胰岛素原在CFPS系统中的可溶性表达,其融合荧光蛋白的策略显著提升了胰岛素原的可溶性,该结果为探究胰岛素合成新方法及开发基于CFPS系统的新型胰岛素给药途径奠定了基础。     

Control of protein synthesis in a wheat germ cell-free system

Thermostable low molecular weight translational inhibitor was found in wheat germ cell-free extract. The inhibitor was formed during preincubation of wheat S-23 fraction with components of the energy-supplying system (ATP, GTP, phosphoenolpyruvate) in the absence of exogenous mRNA. The inhibitor effectively and irreversibly blocks protein synthesis in both wheat germ and rabbit reticulocyte systems. Our results seem to suggest that the inhibitor can activate wheat endogenous mRNA, which under the standard conditions does not reveal template activity but, once activated, can effectively compete with exogenous mRNA.     

Development of a rapid and productive cell-free protein synthesis system

Due to recent advances in genome sequencing, there has been a dramatic increase in the quantity of genetic information, which has lead to an even greater demand for a faster, more parallel expression system. Therefore, interest in cell-free protein synthesis, as an alternative method for high-throughput gene expression, has been revived. In contrast toin vivo gene expression methods, cell-free protein synthesis provides a completely open system for direct access to the reaction conditions. We have developed an efficient cell-free protein synthesis system by optimizing the energy source and S30 extract. Under the optimized conditions, approximately 650 μg/mL of protein was produced after 2 h of incubation, with the developed system further modified for the efficient expression of PCR-amplified DNA. When the concentrations of DNA, magnesium, and amino acids were optimized for the production of PCR-based cell-free protein synthesis, the protein yield was comparable to that from the plasmid template.     

Characterization of a protein synthesis system from rat liver. Translation of endogenous and exogenous messenger RNA

An in vitro system prepared from rat liver postmitochondrial supernatant exhibits a high rate of protein synthesis for an extended period of time. This system initiates translation of either endogenous or exogenous mRNA, incorporates Met at a rate of 13 pmol/mg of postmitochondrial supernatant protein/min, maintains this rate for at least 90 min, and performs several rounds of translation/mRNA molecule. Up to 50% of the activity is due to reinitiation of protein synthesis using endogenous mRNA. In addition, 60-70% of the protein synthesized was released from ribosomes into the medium. Addition of globin mRNA stimulates protein synthesis and results in the synthesis of a protein that comigrates with authentic rabbit globin. Black beetle virus mRNA 2 also stimulates protein synthesis and results in synthesis of a protein with molecular weight corresponding to that of the mature viral protein. With endogenous rat liver mRNA this system synthesizes a large number of proteins.     

Translation of honeybee promelittin messenger RNA. Formation of a larger product in a mammalian cell-free system.

Venom glands of honeybees synthesize the peptide melittin via the precursor promelittin. Total RNA preparations from venom glands served as template in a cell-free system prepared from mammalian cells. The heterologous system translated the insect mRNA with approximately the same efficiency as hemoglobin mRNA. A polypeptide was synthesized which, as shown by acrylamide gel electrophoresis in the presence of detergent, has a higher molecular weight than promelittin. Analysis of peptic fragments as well as Edman degradation have demonstrated that sequences characteristic of venom gland promelittin are present in this product formed in vitro. Furthermore, a bacterial protease which specifically splits after acidic residues liberates from the cell-free product a fragment which closely resembles melittin. Evidence is presented that most of the extra amino acids are located at the amino terminus of the product formed in vitro. The larger polypeptide detected in vitro may represent a precursor of promelittin.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.