Rapid preparation of human insulin and insulin analogues in high yield by enzyme-assisted semi-synthesis.
Ontology highlight
ABSTRACT: Reaction conditions are described that permit the enzyme-assisted semi-synthetic replacement of residue B30 of pig insulin (or of analogue) to proceed in very high yield in 2 h or less. Immobilized trypsin may be used as catalyst, and excess amino acid ester may be recycled after a simple extraction. Alanine-B30 may be replaced by a variety of nucleophiles, including threonine O-t-butyl ether t-butyl ester, in which case the yield of crude product is about 99%. De-protection of the B30 threonyl ester analogue of insulin thus formed then affords human insulin in an overall yield of about 92%, based on pig starting material. The product has full biological potency, as determined by depression of blood glucose concentration in rats, and showed the expected behaviour on radioimmunoassay.
Project description:One of the central goals for the development of cell-free (CF) gene expression systems is to ensure reproducible expression of proteins at high quality and yield. Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols have been developed and optimized reaction conditions have been established. One of the crucial steps during the preparation of cell extract-based expression systems, however, is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here, we demonstrate for an E. coli based system that a lysis procedure combining incubation of the cells with lysozyme with a gentle sonication step results in highly active cell extracts. As examples for the capabilities of our extract, we demonstrate the production of several fluorescent proteins as well as the production and assembly of T7 bacteriophages. Compared to other cell-free expression systems, our method achieved the highest phage titers in our plaque assays. Stateof-the-art quantitative proteomics revealed that enzymes of the energy regeneration pathway were enriched in our extract compared to a widely used commercial cell extract. On the other hand, ribosomal proteins were found to be more abundant in the commercial product.
Project description:The evergrowing plastic production and the caused concerns of plastic waste accumulation have stimulated the need for waste plastic chemical recycling/valorization. Current methods suffer from harsh reaction conditions and long reaction time. Herein we demonstrate a non-thermal plasma-assisted method for rapid hydrogenolysis of polystyrene (PS) at ambient temperature and atmospheric pressure, generating high yield (>40 wt%) of C1-C3 hydrocarbons and ethylene being the dominant gas product (Selectivity of ethylene, SC2H4 > 70%) within ~10 min. The fast reaction kinetics is attributed to highly active hydrogen plasma, which can effectively break bonds in polymer and initiate hydrogenolysis under mild condition. Efficient hydrogenolysis of post-consumer PS materials using this method is also demonstrated, suggesting a promising approach for fast retrieval of small molecular hydrocarbon modules from plastic materials as well as a good capability to process waste plastics in complicated conditions.
Project description:The synthesis of nanosized metal-organic frameworks (NMOFs) is requisite for their application as injectable drug delivery systems (DDSs) and other biorelevant purposes. Herein, we have critically examined the role of different synthetic parameters leading to the production of UiO-66 crystals smaller than 100 nm. Of note, we demonstrate the co-modulator role conferred by halide ions, not only to produce NMOFs with precise morphology and size, but also to significantly improve the reaction yield. The resulting NMOFs are highly crystalline and exhibit sustained colloidal stability in different biologically relevant media. As a proof of concept, these NMOFs were loaded with Rhodamine 6G (R6G), which remained trapped in most common biologically relevant media. When incubated with living mammalian cells, the R6G-loaded NMOFs were efficiently internalized and did not impair cell viability even at relatively high doses.
Project description:The classical synthesis of quinoids, which involves Takahashi coupling and subsequent oxidation, often gives only low to medium yields. Herein, we disclose the keto-enol-tautomerism-assisted spontaneous air oxidation of the coupling products to quinoids. This allows for the synthesis of various indandione-terminated quinoids in high isolated yields (85-95%). The origin of the high yield and the mechanism of the spontaneous air oxidation were ascertained by experiments and theoretical calculations. All the quinoidal compounds displayed unipolar n-type transport behavior, and single crystal field-effect transistors based on the micro-wires of a representative quinoid delivered an electron mobility of up to 0.53 cm2 V-1 s-1, showing the potential of this type of quinoid as an organic semiconductor.
Project description:A microwave enhanced synthesis of prodrug nucleotide (ProTide) analogues is presented. Comparison of conventional thermal heating reaction with microwave irradiation exemplifies the potential of the novel methodology herein presented for the selective 5'-phosphoramidate synthesis, without protection of the 3' position in the ribonucleoside.
Project description:Complete enzymatic digestion of proteins for bottom-up proteomics is substantially improved by use of detergents for denaturation and solubilization. Detergents however, are incompatible with many proteases and highly detrimental to LC-MS/MS. Recently; filter-based methods have seen wide use due to their capacity to remove detergents and harmful reagents prior to digestion and mass spectrometric analysis. We hypothesized that non-specific protein binding to negatively charged silica-based filters would be enhanced by addition of lyotropic salts, similar to DNA purification. We sought to exploit these interactions and investigate if low-cost DNA purification spin-filters, 'Minipreps,' efficiently and reproducibly bind proteins for digestion and LC-MS/MS analysis. We propose a new method, Miniprep Assisted Proteomics (MAP), for sample preparation. We demonstrate binding capacity, performance, recovery and identification rates for proteins and whole-cell lysates using MAP. MAP recovered equivalent or greater protein yields from 0.5-50 μg analyses benchmarked against commercial trapping preparations. Nano UHPLC-MS/MS proteome profiling of lysates of Escherichia coli had 99.3% overlap vs. existing approaches and reproducibility of replicate minipreps was 98.8% at the 1% FDR protein level. Label Free Quantitative proteomics was performed and 91.2% of quantified proteins had a %CV <20% (2044/2241). Miniprep Assisted Proteomics can be performed in minutes, shows low variability, high recovery and proteome depth. This suggests a significant role for adventitious binding in developing new proteomics sample preparation techniques. MAP represents an efficient, ultra-low-cost alternative for sample preparation in a commercially obtainable device that costs ∼$0.50 (USD) per miniprep.
Project description:Hsp90 is an attractive therapeutic target for the treatment of cancer. Extensive structural modifications to novobiocin, the first Hsp90 C-terminal inhibitor discovered, have produced a library of novobiocin analogues and revealed some structure-activity relationships. Based upon the most potent novobiocin analogues generated from prior studies, a three-dimensional quantitative structure-activity (3D-QSAR) model was built. In addition, a new set of novobiocin analogues containing various structural features supported by the 3D-QSAR model were synthesized and evaluated against two breast cancer cell lines. Several new inhibitors produced anti-proliferative activity at mid nano-molar concentrations, which results through Hsp90 inhibition.
Project description:The development of luminescent materials greatly affects the development of fluorescence imaging technology. The preparation of carbon dots (CDs) with high photoluminescence quantum yield (PLQY) in the solid-state is challenging due to excessive resonance energy transfer (RET) and direct π-π interactions. In this study, we synthesized carbon dots that exhibit green fluorescence (GCDs) with absolute PLQYs up to 35.65% in one step by a microwave-assisted method. In the solid-state, the absolute PLQY reached 19.25%. Then, the GCDs were mixed with soluble starch in appropriate proportions, which improved the adsorption and dispersion of the GCDs and greatly reduced the cost of the fingerprint powder, and increased the absolute PLQY of the fingerprint powder to 41.75%. Finally, we prepared GCDs for preliminary fabrication of luminescent films, and the GCD-starch powder was successfully applied to high-quality latent fingerprint (LFP) imaging. The related properties of GCDs and the LFP detection performance of fingerprint detection powders prepared by GCDs were studied in detail. The results showed that the LFP system developed with GCDs-starch powder visualized LFPs with high definition and contrast under different conditions, and GCDs had potential for application in light-emitting devices. This study developed a new type of solid-state luminescent CDs and demonstrated that these GCDs have great application potential for LFP detection. This study may also provide inspiration for other applications based on efficient solid-state fluorescence.
Project description:Botulinum neurotoxins (BoNTs) are the most toxic proteins known to cause flaccid muscle paralysis as a result of inhibition of neurotransmitter release from peripheral cholinergic synapses. BoNT type A (BoNT/A) is a 150 kDa protein consisting of two major subunits: light chain (LC) and heavy chain (HC). The LC is required for the catalytic activity of neurotoxin, whereas the C and N terminal domains of the HC are required for cell binding, and translocation of LC across the endosome membranes, respectively. To better understand the structural and functional aspects of BoNT/A intoxication we report here the development of high yield Escherichia coli expression system (2-20-fold higher yield than the value reported in the literature) for the production of recombinant light chain-translocation domain (rLC-TD/A) module of BoNT/A which is catalytically active and translocation competent. The open reading frame of rLC-TD/A was PCR amplified from deactivated recombinant BoNT/A gene (a non-select agent reagent), and was cloned using pET45b (+) vector to express in E. coli cells. The purification procedure included a sequential order of affinity chromatography, trypsinization, and anion exchange column chromatography. We were able to purify > 95% pure, catalytically active and structurally well-folded protein. Comparison of enzyme kinetics of purified LC-TD/A to full-length toxin and recombinant light chain A suggest that the affinity for the substrate is in between endopeptidase domain and botulinum toxin. The potential application of the purified protein has been discussed in toxicity and translocation assays.
Project description:Antivenoms from hyperimmune animal plasma are the only specific pharmaceuticals against snakebites. The improvement of downstream processing strategies is of great interest, not only in terms of purity profile, but also from yield-to-cost perspective and rational use of plasma of animal origin. We report on development of an efficient refinement strategy for F(ab')2-based antivenom preparation. Process design was driven by the imperative to keep the active principle constantly in solution as a precautionary measure to preserve stability of its conformation (precipitation of active principle or its adsorption to chromatographic stationary phase has been completely avoided). IgG was extracted from hyperimmune horse plasma by 2% (V/V) caprylic acid, depleted from traces of precipitating agent and digested by pepsin. Balance between incomplete IgG fraction breakdown, F(ab')2 over-digestion and loss of the active principle's protective efficacy was achieved by adjusting pepsin to substrate ratio at the value of 4:300 (w/w), setting pH to 3.2 and incubation period to 1.5 h. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. Developed manufacturing strategy gave 100% pure and aggregate-free F(ab')2 preparation, as shown by size-exclusion HPLC and confirmed by MS/MS. The overall yield of 75% or higher compares favorably to others so far reported. This optimised procedure looks also promising for large-scale production of therapeutic antivenoms, since high yield of the active drug and fulfillment of the regulatory demand considering purity was achieved. The recovery of the active substance was precisely determined in each purification step enabling accurate estimation of the process cost-effectiveness.