Total amino acid stabilization during cell-free protein synthesis reactions

Limitations in amino acid supply have been recognized as a substantial problem in cell-free protein synthesis reactions. Although enzymatic inhibitors and fed-batch techniques have been beneficial, the most robust way to stabilize amino acids is to remove the responsible enzymatic activities by genetically modifying the source strain used for cell extract preparation. Previous work showed this was possible for arginine, serine, and tryptophan, but cysteine degradation remained a major limitation in obtaining high protein synthesis yields. Through radiolabel techniques, we confirmed that cysteine degradation was caused by the activity of glutamate-cysteine ligase (gene gshA) in the cell extract. Next, we created Escherichia coli strain KC6 that combines a gshA deletion with previously described deletions for arginine, serine, and tryptophan stabilization. Strain KC6 grows well, and active cell extract can be produced from it for cell-free protein synthesis reactions. The extract from strain KC6 maintains stable amino acid concentrations of all 20 amino acids in a 3-h batch reaction. Yields for three different proteins improved 75-250% relative to cell-free expression using the control extract.     

Activity of taraxasteryl acetate on inflammation and heat shock protein synthesis

Pluchea sagittalis whole plant dichloromethane extract showed inhibitory activity in several inflammatory models: rat hind paw-edema, mice ear edema, and air-pouch rat granuloma. The extract inhibited the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in stimulated human neutrophils. It also showed inhibitory effect on heat shock protein 72 (hsp72) synthesis in stimulated neutrophils, while it had opposite effects on unstimulated cells. The triterpene taraxasteryl acetate was obtained from the dichloromethane extract by bioassay directed isolation, being active against induced ROS and RNS production in human neutrophils. In mice ear edema (induced by phorbol-12-mirystate-13-acetate, croton oil and arachidonic acid), taraxasteryl acetate showed a topical anti-inflammatory activity similar to the extract, but at 1/20 of the dose. The same ratio was observed for the inhibition of hsp72 production in stimulated human neutrophils. In unstimulated monocytes and neutrophils, taraxasteryl acetate showed a higher stimulating activity of hsp72 production than the extract, involving different mechanisms in each cell type. To our knowledge, taraxasteryl acetate is the first natural product for which a dual effect on the hsp response is reported.     

Cell-free synthesis of encephalomyocarditis virus

We developed a system for complete replication of encephalomyocarditis virus (EMCV) in a test tube by using an in vitro translation extract from Krebs-2 cells. Efficient virus synthesis occurred in a narrow range of Mg(2+) and EMCV RNA concentrations. Excess input RNA impaired RNA replication and virus production but not translation. This suggests the existence of a negative-feedback mechanism for regulation of RNA replication by the viral plus-strand RNA or proteins.     

Biomediated synthesis of silver nanoparticles using Exiguobacterium mexicanum

Biomediated silver nanoparticle were synthesized using a cell free extract of a soil bacterium, Exiguobacterium mexicanum PR 10.6. The silver nanoparticles were characterised using UV–Vis spectroscopy, energy dispersive spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy. The nanoparticles ranged from 5 to 40 nm. Extracellular polymeric substance played a critical role in the reduction of silver ion and nanoparticle stabilisation when using the cell free extract. The synthesis using E. mexicanum is an effective eco-friendly, rapid method for silver nanoparticle synthesis within 1 h.     

Effects of growth rate on cell extract performance in cell-free protein synthesis

Cell-free protein synthesis is a useful research tool and now stands poised to compete with in vivo expression for commercial production of proteins. However, both the extract preparation and protein synthesis procedures must be scaled up. A key challenge is producing the required amount of biomass that also results in highly active cell-free extracts. In this work, we show that the growth rate of the culture dramatically affects extract performance. Extracts prepared from cultures with a specific growth rate of 0.7/h or higher produced approximately 0.9 mg/mL of chloramphenicol acetyl transferase (CAT) in a batch reaction. In contrast, when the source culture growth rate was 0.3/h, the resulting extract produced only 0.5 mg/mL CAT. Examination of the ribosome content in the extracts revealed that the growth rate of the source cells strongly influenced the final ribosome concentration. Polysome analysis of cell-free protein synthesis reactions indicated that about 22% of the total 70S ribosomes are in polysomes for all extracts regardless of growth rate. Furthermore, the overall specific production from the 70S ribosomes is about 22 CAT proteins per ribosome over the course of the reaction in all cases. It appears that rapid culture growth rates are essential for producing a productive extract. However, growth rate does not seem to influence specific ribosome activity. Rather, the increase in extract productivity is a result of a higher ribosome concentration. These results are important for cell-free technology and also suggest an assay for intrinsic in vivo protein synthesis activity.     

Optimization of ectoine synthesis through fed-batch fermentation of Brevibacterium epidermis

A production process for ectoine has been developed, using Brevibacterium epidermis DSM20659 as the producer strain. First, the optimal conditions for intracellular synthesis of ectoine were determined. The size of the intracellular ectoine pool is shown to be dependent on the external salt concentration, type of carbon source, and yeast extract concentration. Under the optimized conditions of 1 M NaCl, 50 g/L monosodium glutamate, and 2.5 g/L yeast extract, a maximum concentration of intracellular ectoine of 0.9 g/L was obtained in shake flask cultures. After optimizing the batch fermentation parameters of temperature, pH, agitation, and aeration, the yield could be further increased by applying the fed-batch fermentation principle in 1.5- to 2-L fermentors. Glutamate and yeast extract were fed to the bacterial cells such that the total glutamate concentration in the broth remained constant. A total yield of 8 g ectoine/L fermentation broth was obtained with a productivity of 2 g ectoine/L/day. After the bacterial cells were harvested from the culture broth, the ectoine was recovered from them by a two-step extraction with water and ethanol. Crystallization of the product was obtained after concentration of the extract via evaporation under reduced pressure. After this downstream process, 55% of the ectoine produced in the fermentor could be crystallized in four fractions. The first fractions were of very high purity (98%). This production process can compete with other described production processes for ectoine in productivity and simplicity. Further advantages are the relatively low amounts of NaCl needed and the absence of hydroxyectoine, often a byproduct, in the final product.     

Ecteinascidia turbinata extracts inhibit DNA synthesis in lymphocytes after mitogenic stimulation by lectins.

Aqueous ethanol extract of a tunicate which was previously found to exert antitumor and immunosuppressive activities in vivo was tested for its effect on normal human lymphocytes in vitro. The extract suppressed the uptake of tritiated thymidine by lymphocytes stimulated with mitogen. This suppressive effect did not require continuous presence of the extract. Treatment of lymphocytes prior to mitogenic stimulation resulted in suppressive effect. The fact that suppression by the extract could also be achieved 24 hr after exposure to mitogen, an interval which was found to suffice for the attainment of maximal commitment for blastogenic transformation indicates that Ete can act at a stage subsequent to the binding of the lectin and elicitation of a mitogenic signal(s).     

Cell-free synthesis of the dipeptide antibiotic bacilysin

Summary Bacilysin, a dipeptide antibiotic produced byBacillus subtilis A 14, was synthesized by a cell-free extract of the producing organism from its constitutent amino acids,l-alanine andl-anticapsin. The synthesis required ATP and Mg²⁺ and was optimal at pH 8.1. The same extract also synthesizedl-alanyl-l-alanine. The synthesis of bacilysin was not inhibited by chloramphenicol, DNase or RNase.     

Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system

In this study, as a part of our efforts to improve the robustness and economical feasibility of cell-free protein synthesis, we developed a simple method of preparing the cell extracts used for catalyzing cell-free protein synthesis reactions. We found that the high-speed centrifugation, pre-incubation, and dialysis steps of the conventional procedures could be omitted without losing the translational activity of the resulting cell extract. Instead, a simple centrifugation step at low speed (12,000 RCF for 10 min) followed by a brief period of incubation was sufficient for the preparation of an active extract to support cell-free protein synthesis with higher productivity and consistency. Compared to the present standard procedures for the preparation of the S30 extract, the overall cost of the reagents and processing time were reduced by 80 and 60%, respectively.     

Interaction between membrane functions and protein synthesis in reticulocytes. A membrane constituent which inhibits protein synthesis in cell-free system.

The reticulocyte cell membrane was investigated for the role that it plays in cellular protein synthesis. It was found that a strong inhibition in protein synthesis occurred when a membranal extract was added to a cell-free protein-synthesizing system. The membranal extract was obtained by disrupting and then sulubilizing cell membranes with the non-ionic detergent Triton X-100. The inhibition in protein synthesis could neither be attributed to an RNAse-type action nor to a reduction in the energy level of the system. Preliminary results show that the principal site of action occurs at the elongation or/and termination stages of protein synthesis. Initial purification of the inhibitory component was achieved.     

Optimization of some variables that affect the synthesis of laccase by Pleurotus ostreatus

The influence of initial pH, concentration of yeast extract, inducer, type of enzyme releaser and buffer system on the composition of a medium for laccase production by Pleurotus ostreatus DM-1513 was investigated. A 2⁵ full factorial experimental design was initially employed to evaluate the effects of these variables on the enzyme synthesis. Data analysis showed that low pH and high yeast extract concentration values, as well as the absence of both an inducer and a buffer system, had positive effects on the secreted enzyme levels, whereas the type of enzyme releaser did not have a significant effect. The highest levels of laccase activity (489–540?U/l) were obtained in optimization experiments using media with initial pH between 6.0 and 6.5 and yeast extract concentrations of 0–0.25%     

Biochemical estimation of Moringa oleifera leaf extract for synthesis of silver nanoparticle mediated drug delivery system

Breast tissue remodeling occurs throughout a women’s life from pre-menstrual to post menopausal time, hence there is a high risk of DNA damage. Estrogen metabolite generates free radical that causes DNA damage leading to cancer cell formation. Moringa oleifera leaf extract contain rich amount of Polyphenolic and Flavonoids compound which trap the free radical and thereby prevent cancer cell progression. Moreover anti-gonadotrophic hormonal activity of the leaf extract slows down the proliferation rate of T47D cell line. Hence our aim of study mainly focused to develop an anticancer drug delivery system using M. oleifera leaf extract. Silver nanoparticle (AgNp) is formed by green synthesis method using M. oleifera leaf extract. Reducing sugar, Flavonoids, Polyphenolic compound, and NADPH dependent dehydrogenase present in M. oleifera leaf extract are responsible for reduction of the silver ion (Ag+) followed by formation of metallic silver (Ag0). The formation of spherical and rectangular shaped AgNp with average size of 50 nm is reflected with sharp SPR band near 420 nm and from SEM, TEM analysis. The nanoparticle mediated polyvinyl alcohol composite film (nano-rod) is prepared for delivery of the purified anti-neoplastic phyto-compound from leaf extract. The large surface area of AgNp permits its coordination with purified compound molecule by metal ligand bonding. Therefore it provide duel effect, it acts as carrier molecule for phyto-compound and also creates cytotoxicity effect on cancer cells. Single dose treatment on HeLa cell indicates that the composite film is more effective in killing the cancer cell than the purified anti-neoplastic phyto-compound.     

Effect of saffron on cell colony formation and cellular nucleic acid and protein synthesis.

A concentrated extract of saffron was prepared from the flowers of Crocus sativis. The effect of this extract on the ability of HeLa cells to form colonies, and on cellular DNA, RNA and protein synthesis was examined. Incubation of cells with extract for 3 h resulted in significant inhibition of colony formation and cellular nucleic acid synthesis with 50% inhibition at concentrations of approximately 100-150 micrograms/ml. In contrast there was no inhibition of cellular protein synthesis at concentrations of extract as high as 400 micrograms/ml.     

Circadian variations in pyrimidine nucleotide synthesis in rat liver

The biosynthesis of pyrimidine components in rat liver varies with the time of the day. The concentrations of both the cytidine and the uridine components of the acid-soluble extract are lowest in the morning hours and highest around midnight. The utilization of [2-¹⁴C]orotic acid for the synthesis of the pyrimidine components of the acid-soluble extract, RNA, and DNA has a similar character. Analogous changes also are seen in the uptake of [U-¹⁴C]cytidine and its utilization for the synthesis of RNA cytosine.     

Green synthesis of gold nanoparticles with Zingiber officinale extract: Characterization and blood compatibility

Biosynthesis of gold nanoparticles with small size and biostability is very important and used in various biomedical applications. There are lot of reports for the synthesis of gold nanoparticles by the addition of reducing agent and stabilizing agent. In the present study we have synthesized gold nanoparticles, with a particle size ranging from 5 to 15 nm, using Zingiber officinale extract which acts both as reducing and stabilizing agent. Z. officinale extract is reported to be a more potent anti-platelet agent than aspirin. Therefore, green synthesis of gold nanoparticles with Z. officinale extract, as an alternative to chemical synthesis, is beneficial from its biological and medical applications point of view, because of its good blood biocompatibility and physiological stability. The formation and size distribution of gold nanoparticles were confirmed by dynamic light scattering (DLS), UV–vis spectrophotometer and transmission electron microscopy (TEM). Gold nanoparticles synthesized using citrate and Z. officinale extract demonstrated very low protein adsorption. Both nanoparticles were non platelet activating and non complement activating on contact with whole human blood. They also did not aggregate other blood cells, however, nanoparticles synthesised with Z. officinale extract was highly stable at physiological condition compared to citrate capped nanoparticles, which aggregated. Thus the usage of nanoparticles, synthesized with Z. officinale extract, as vectors for the applications in drug delivery, gene delivery or as biosensors, where a direct contact with blood occurs is justified.     

On the mechanism of action of free purine bases on DNA synthesis in serum-starved L-cells treated with platelet extract

It has previously been found that there is a synergistic effect of free purine bases and low concentrations of dialyzed platelet extract on net synthesis of DNA in serum-starved fibroblast-like mouse L-cells. Experiments with a mutant line of L-cells that was deficient in hypoxanthine phosphoribosyl transferase (EC 2.4.2.8) indicated that purine bases had a stimulatory effect only if they were incorporated into cellular ribonucleotides. In the present paper it was shown that platelet extract induced the incorporation of hypoxanthine or adenine into both ATP and DNA. The induced net synthesis of DNA appears to take place in the nuclei and it requires that platelet extract is present in the medium only initially while free purine bases have to be present only later in the period of the experiment when DNA is being synthesized. The induction of both incorporation of free purine bases into DNA and ATP and of net DNA synthesis is dependent on heat-labile components in platelet extract. The extract cannot be substituted for by platelet derived growth factor.     

Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing

Escherichia coli-based cell extract is a vital component of inexpensive and high-yielding cell-free protein synthesis reactions. However, effective preparation of E. coli cell extract is limited to high-pressure (French press-style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. Here we report the viability of E. coli cell extract prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples. Specifically, we assessed the low-capital-cost lysis techniques of: (i) sonication, (ii) bead vortex mixing, (iii) freeze-thaw cycling, and (iv) lysozyme incubation to prepare E. coli cell extract for cell-free protein synthesis (CFPS). We also used simple shake flask fermentations with a commercially available E. coli strain. In addition, RNA polymerase was overexpressed in the E. coli cells prior to lysis, thus eliminating the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high-yielding E. coli-based extract was prepared using equipment requiring a reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing.     

Neural factors that stimulate ecdysteroid synthesis by the larval ring gland ofDrosophila melanogaster

Summary The larval ring gland ofDrosophila melanogaster is the source of ecdysteroids responsible for larval-larval and larval-pupal molting. An extract prepared from theDrosophila larval central nervous system, that presumably contains prothoracicotropic hormone, elicits a significant and dose-dependent in vitro increase in ecdysteroid synthesis by ring glands from wandering third instar larvae. The synthesis of all three ecdysteroids previously identified as ring gland products is elevated by more than two-fold in the presence of neural extract. The maximum response occurs within 30 min and can be sustained for at least two hours after a 30 min exposure to neural extract. No non-neural tissue extracts evoke a response and most of the prothoracicotropic activity originates in the ventral ganglion. However, while extract prepared from larval brains elicits only a slight increase in ecdysteroid synthesis, it enhances the activity of a submaximal dose of ventral ganglion extract. This suggests that two or more neural factors, at least one from the brain lobes and another from the ventral ganglion, interact to stimulate ecdysteroid synthesis by the larval ring gland.Abbreviations CNS central nervous system - HPLC high performance liquid chromatography - PTTH prothoracicotropic hormone - RIA radioimmunoassay     

Initiation of chromosomal DNA synthesis in vitro

Summary An in vitro complementation assay for initiation of chromosomal DNA replication is described. The initiation reaction is dependent upon extract from either of two hybrid-plasmid containing strains. Each hybrid plasmid carries a suppressor of dnaA-ts mutations. The in vitro DNA synthesis is heavily biased toward the origin region, and the origin of replication (oriC) is replicated as determined by DNA-DNA hybridizations.