Coli Cell-free Protein Synthesis Research Articles

De novo expression and antibacterial potential of four lactoferricin peptides in cell-free protein synthesis system

For the first time, we produced four lactoferricin (LFcin) peptides by a cell-free (in vitro) method. These short antimicrobial peptides were expressed in an E. coli cell-free protein synthesis (CFPS) system and the bioactivity of the produced peptides was demonstrated. Additionally, we designed a novel synthetic consensus peptide (ConLFcin). The genes of bovine Lfcin (bLFcin), human Lfcin (hLFcin), camel Lfcin (cLFcin), and ConLFcin were cloned into pET101/D-TOPO vector then peptides were synthesized in vitro by E. coli CFPS system. The antibacterial activity of these synthesized peptides was evaluated against Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). The four cell-free synthesized peptides showed significant antibacterial potency at minimum inhibitory concentration (MIC) values between 1.25 and 10 μg/mL. cLFcin and ConLFcin showed higher antibacterial effects than bLFcin and hLFcin. Thus, cell-free expression system is an ideal system for rapid expression of functionally active short bioactive peptides.

Toward a genome scale sequence specific dynamic model of cell-free protein synthesis in Escherichia coli

In this study, we developed a dynamic mathematical model of E. coli cell-free protein synthesis (CFPS). Model parameters were estimated from a dataset consisting of glucose, organic acids, energy species, amino acids, and protein product, chloramphenicol acetyltransferase (CAT) measurements. The model was successfully trained to simulate these measurements, especially those of the central carbon metabolism. We then used the trained model to evaluate the performance, e.g., the yield and rates of protein production. CAT was produced with an energy efficiency of 12%, suggesting that the process could be further optimized. Reaction group knockouts showed that protein productivity was most sensitive to the oxidative phosphorylation and glycolysis/gluconeogenesis pathways. Amino acid biosynthesis was also important for productivity, while overflow metabolism and TCA cycle affected the overall system state. In addition, translation was more important to productivity than transcription. Finally, CAT production was robust to allosteric control, as were most of the predicted metabolite concentrations; the exceptions to this were the concentrations of succinate and malate, and to a lesser extent pyruvate and acetate, which varied from the measured values when allosteric control was removed. This study is the first to use kinetic modeling to predict dynamic protein production in a cell-free E. coli system, and could provide a foundation for genome scale, dynamic modeling of cell-free E. coli protein synthesis.

Cell-free expression, purification and characterization of Drosophila melanogaster odorant receptor OR42a and its co-receptor

Olfactory receptors (OR), a group of classic membrane proteins, plays a vital role in insect reproduction and acclimatization. Deciphering the molecular mechanism of insect olfaction could enhance pest control and environmental protection. Studies on ORs have faced a major bottleneck due to the notoriously strong hydrophobicity of ORs, which results in difficult expression in heterologous cell systems. Here, we demonstrated that insect ORs could be functionally produced using the E. coli cell-free protein synthesis system (CFPS), in which the highest yield of total ORs is 350 μg per 1 ml reaction. We tested the effects of detergent types and concentrations on soluble expression of ORs. The ORs showed a classic α-helical infrared spectrum. Quartz crystal microbalance (QCM) was used to demonstrate that ORs fold correctly and respond to their ligands. This is the first report that insect OR42a could be functionally produced in vitro. This approach may facilitate the development of biomimetic olfactory biosensors and may also be utilized for drug positioning and development, environmental protection and agriculture.

Rapid Generation of Monoclonal Antibodies from Single B Cells by Ecobody Technology.

Single B cell sampling following to direct gene amplification and transient expression in animal cells has been recognized as powerful monoclonal antibodies (mAbs) screening strategies. Here we report Ecobody technology which allows mAbs screening from single B cells in two days This technology uses Escherichia coli cell-free protein synthesis (CFPS) for mAb expression. In the CFPS step, we employed our original techniques: (1) ‘Zipbody’ as a modified Fab (fragment of antigen binding) format, in which the active Fab formation is facilitated by adhesive leucine zipper peptides fused at the C-termini of the light and heavy chains; and (2) an N-terminal SKIK peptide tag that can markedly increase protein production. By the Ecobody technology, we demonstrated rapid screening of antigen specific mAbs from immunized rabbits and Epstein-Barr Virus infected human B cells. We further obtained rabbit mAbs in E. coli expression system yielding to 8.5 mg of purified proteins from 1 L bacterial culture.

Ecobody technology: rapid monoclonal antibody screening method from single B cells using cell-free protein synthesis for antigen-binding fragment formation

We report a rapid and cost-effective monoclonal antibody screening method from single animal B cells using reverse transcription (RT)-PCR and Escherichia coli cell-free protein synthesis (CFPS), which allows evaluation of antibodies within 2 working days. This process is named “Ecobody technology”. The method includes strategies to isolate B cells that specifically bind an antigen from the peripheral blood of immunised animals, and single-cell RT-PCR to generate DNA fragments of the VH and VL genes, followed by CFPS for production of fragments of antigen binding (Fab). In the CFPS step, we employed our techniques: 1) ‘Zipbody’ as a method for producing Fab, in which the association of heavy and light chains is facilitated by adhesive leucine zipper peptides fused at the C-termini of the Fab; and 2) an N-terminal SKIK peptide tag that can increase protein expression levels. Using Ecobody technology, we obtained highly-specific monoclonal antibodies for the antigens Vibrio parahaemolyticus and E. coli O26. The anti-V. parahaemolyticus Zipbody mAb was further produced in E. coli strain SHuffle T7 Express in inclusion bodies and refolded by a conventional method, resulting in significant antigen-binding activity (KD = 469 pM) and productivity of 8.5 mg purified antibody/L-culture.

Simplifying and streamlining Escherichia coli‐based cell‐free protein synthesis

Escherichia coli cell-free protein synthesis (CFPS) uses E. coli extracts to make active proteins in vitro. The basic CFPS reaction mixture is comprised of four main reagent components: (1) energy source and CFPS chemicals, (2) DNA encoding the protein of interest, (3) T7 RNA Polymerase (RNAP) for transcription, and (4) cell extract for translation. In this work, we have simplified and shortened the protocols for preparing the CFPS chemical mixture, cell extract, and T7 RNAP. First, we streamlined the workflow for preparing the CFPS chemical solutions by combining all the chemicals into a single reagent mixture, which we call Premix. We showed that productive cell extracts could be made from cells grown in simple shake flasks, and we also truncated the preparation protocol. Finally, we discovered that T7 RNAP purification was not necessary for CFPS. Crude lysate from cells over-expressing T7 RNAP could be used without deleteriously affecting protein production. Using chloramphenicol acetyltransferase (CAT) as a model protein, we showed that these streamlined protocols still support high-yielding CFPS. These simplified procedures save time and offer greater accessibility to our laboratory's CFPS technology.

Localization of BiP to translating ribosomes increases soluble accumulation of secreted eukaryotic proteins in an Escherichia coli cell‐free system

The endoplasmic reticulum (ER) resident Hsp70 chaperone, BiP, docks to the Sec translocon and interacts co-translationally with polypeptides entering the ER to encourage proper folding. In order to recreate this interaction in Escherichia coli cell-free protein synthesis (CFPS) reactions, a fusion protein was formed between the ribosome-binding portion of the E. coli protein trigger factor (TF) and BiP. The biophysical affinity to ribosomes as well as the characteristic Hsp70 ATPase activity were both verified for the fusion protein. When added to E. coli-based CFPS reactions, the TF-BiP fusion chaperone increased soluble yields of several protein fragments that are normally secreted through the ER and have poor solubility in typical CFPS reactions. For comparison, a fusion between TF and the native E. coli Hsp70, DnaK, was also constructed. This fusion was also biologically active and increased soluble yields of certain protein targets in CFPS. The TF-BiP fusion described in this study can be seen as a first step in reconstituting and better understanding ER folding pathways in the prokaryotic environment of E. coli CFPS.

Enhancement of protein synthesis by an inorganic polyphosphate in an E. coli cell-free system

In an E. coli cell-free protein synthesis system, the addition of an inorganic polyphosphate [poly(P)] with polyphosphate:AMP phosphotransferase (PAP), which regenerates AMP to ADP, increased the amount of protein synthesis. The maximum yield of the translation product (green fluorescent protein) in the E. coli cell-free system provided by Roche Diagnostics (RTS-100) was 1.16 mg/ml under the optimum reaction condition, which corresponded to a 5.7-fold of that obtained under the standard reaction condition described in the manufacturer's protocol. Interestingly, poly(P) alone enhanced protein synthesis to some extent. When we added poly(P) to the reaction mixture, ATP was consumed at a faster rate, leading to a rapid accumulation of AMP. By adding both poly(P) and PAP to the reaction mixture, an efficient ATP regeneration reaction derived from AMP occurred and the ATP level was recovered. Since the protein synthesis enhancement by poly(P) was also observed when mRNA was added as the template in the reaction, poly(P) accelerated the translation reaction by directly affecting the translation machinery. This also occurred when we used the Pure-system Classic Mini kit (Post Genome Institute) that contained the minimum requirements (pure enzymes and chemicals) for translation and transcription. We also observed that poly(P) extended the half-life of the mRNA template.

Expression of Active Murine Granulocyte-Macrophage Colony-Stimulating Factor in an Escherichia coli Cell-Free System

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.

Modification of polyuridylic acid by bisulfite: Effect on double helix formation and coding properties

Polyuridylic acid was allowed to react with 1M sodium bisulfite at 37°C, pH 7, to produce modified polymers in which up to 95% of the uracil residues were converted to 5,6-dihydrouracil-6-sulfonate residues. Partial saturation of polyuridylic acid by bisulfite sharply reduced its ability to form a helical complex with polyadenylic acid, as measured by melting transition and hypochromicity studies. A 31% saturated polyuridylic acid sample did not interact with polyadenylic acid. The ability of polyuridylic acid to code for phenylalanine incorporation in the E. coli cell free protein synthesis system was very markedly diminished by small degrees of bisulfite modification. Reaction to the extent of 2.6% abolished 46% of the phenylalanine coding ability, for example. This indicates that inactivation of messenger RNA may be one pathway by which bisulfite inflicts biological damage.