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.     

Production of an enzymatic active protein using a continuous flow cell-free translation system.

We have examined the characteristics of protein synthesis in an improved continuous flow cell-free translation system prepared from wheat germ extract with dihydrofolate reductase (dhfr) mRNA as the translated message. Continuous buffer flow and separation of product from the reaction mixture were accomplished by the use of a modified Amicon ultrafiltration chamber as reaction vessel. The system produced protein for more than 20 h, and the product had an activity of dhfr comparable to that of authentic enzyme from E. coli. Analysis of RNA recovered from the filtrate supports the notion that a functionally active protein-synthesizing machinery is superorganized in a dynamic complex.     

A cell-free protein synthesis system as an investigational tool for the translation stop processes

Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.     

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.     

Inhibition of protein synthesis by acetyl-coenzyme A in a cell-free system: possible involvement of protein acetylation in the regulation of translation

Acetyl-coenzyme A (CoASAc) inhibits the rate of incorporation of amino acid into protein in a cell-free system of mouse liver. The effect is more pronounced when exogenous mRNA (tobacco mosaic virus or globin mRNA) rather than endogenous messages are used. Micromolar concentrations of the cofactor block initiation, while millimolar concentrations cause a more general inhibition of the translation process, that affects, in addition, the elongation step. Inclusion of [1-14C]acetyl-CoA in a protein synthesis reaction mixture results in a very rapid and selective labelling of a protein of 200 kd of the 'pH 5' fraction. The possible involvement of the acetylating event in the regulation of protein synthesis is discussed.     

Human transcortin synthesis by a cell-free translation of hepatic mRNA

To evaluate the site of synthesis and to characterize the translated transcortin, poly (A)-containing RNA (mRNA) from human liver was translated in a cell-free system derived from rabbit reticulocyte lysate. The in vitro synthesized product was identified as transcortin by immuno-precipitation with its specific antiserum. This translated transcortin could be displaced from the antibody by unlabeled purified transcortin obtained from plasma. Furthermore, when the translation mixture was applied to a cortisol-Sepharose column, the translated transcortin was bound to the matrix in a specific manner, indicating that this product binds to cortisol. The molecular weight of the translated transcortin was estimated to be 45,700 by its mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis, while that of plasma transcortin was 53,800. The difference in molecular weight between the translated transcortin and plasma transcortin was probably due to the presence of pre-sequence (signal peptide) in addition to the absence of carbohydrate moiety in the former. In conclusion, human liver mRNA directed the synthesis of transcortin, and the translated transcortin binds to cortisol in spite of the absence of carbohydrate moiety.     

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.     

Synthesis and maturation of green fluorescent protein in a cell-free translation system

Summary A cell-free translation system producing mature green fluorescent protein (GFP) can be a useful tool for studying the mechanism and kinetics of GFP chromophore formation, as well as for fast protein engineering. We report here that the mature GFP can be formed in the cell-free translation system from E.coli. The synthesis of GFP in the cell-free system reaches a plateau in 30 to 40 min whereas its maturation is completed in 4 h from the beginning of translation. The delay between the GFP synthesis and the chromophore formation in the cell-free system provides the possibility to isolate and to analyse maturation intermediates for elucidation of the modification pathway.     

Messenger ribonucleic acids from pig intestinal mucosa direct synthesis of calcium-binding protein in a cell-free translation system.

mRNA from pig duodenal mucosa directs synthesis, in a wheat-germ cell-free system, of two products precipitated by antiserum to pure calcium-binding protein. One of these products has a higher molecular weight than authentic calcium-binding protein, but, like the authentic protein, is heat-stable. The other protein co-migrates with pure calcium-binding protein on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, is stable on heating to 70 degrees C for 15 min and alters its elution position on ion-exchange chromatography depending on whether Ca2+ is present in or absent from the elution buffer. The synthesized protein has these properties in common with authentic calcium-binding protein.     

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.     

Multi-hour translation of mRNA in a cell-free system

In this study, we demonstrate that mRNA molecules can serve as an efficient template for cell-free translation through a combination of methods to protect them from nucleolytic digestion. Removal of major endonucleases activity from cell extract, the addition of a stemloop structure at the 3??-end of the mRNA and continuous reloading of ribosomes onto mRNA were found to be crucial for maintaining the functional integrity of mRNA during cell-free synthesis. When these three approaches were combined, mRNA-directed protein synthesis continued over 15 h, leading to the production of 2.6 mg/mL of encoded protein. The methods for direct translation of mRNA presented herein will provide a useful option for deciphering genetic information, including the fields of mRNA display and materialization of metagenomic information.