The site-specific incorporation of non-canonical amino acids (ncAAs) into proteins via amber suppression provides access to novel protein properties, structures, and functions. Historically, poor ...protein expression yields resulting from release factor 1 (RF1) competition has limited this technology. To address this limitation, we develop a high-yield, one-pot cell-free platform for synthesizing proteins bearing ncAAs based on genomically recoded Escherichia coli lacking RF1. A key feature of this platform is the independence on the addition of purified T7 DNA-directed RNA polymerase (T7RNAP) to catalyze transcription. Extracts derived from our final strain demonstrate high productivity, synthesizing 2.67 ± 0.06 g/L superfolder GFP in batch mode without supplementation of purified T7RNAP. Using an optimized one-pot platform, we demonstrate multi-site incorporation of the ncAA p-acetyl-L-phenylalanine into an elastin-like polypeptide with high accuracy of incorporation and yield. Our work has implications for chemical and synthetic biology.
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•A one-pot, high-yield CFPS system was developed from a recoded strain of E. coli•Genomic edits improved the function of a T7 RNA polymerase expressed in the strain•Lysates prepared from the strain function without exogenous biological components•The platform is capable of incorporating up to 40 non-canonical amino acids per protein
Des Soye et al. create and optimize a strain of Escherichia coli that expresses T7 RNA polymerase so that lysates prepared from the strain are enriched with sufficient polymerase to catalyze high-yielding cell-free transcription and translation reactions. Using the resulting platform, the authors synthesize products containing up to 40 non-canonical amino acids.
•A new nanostructured TiO2 film deposited on a stainless steel mesh was prepared.•The new supported photocatalyst was successfully reused several times.•Identified by-products revealed minor ...differences between photocatalytic processes.•Toxicity tests showed that few of them are useful for investigating CECs degradation.
A new supported catalyst composed of a nanostructured TiO2 film deposited on a stainless steel mesh (nanoTiO2-SS) using the Metal Organic Chemical Vapour Deposition (MOCVD) technique was evaluated for the photocatalytic degradation of a mixture of contaminants of emerging concern. Results showed that under the oxidative conditions tested, the nanoTiO2-SS catalyst demonstrated an efficiency in degrading the target contaminants higher than that observed under direct photolysis and photocatalysis using the conventional TiO2 Degussa P25 catalyst at the same amount of TiO2 participating to the photocatalysis. Specifically, the rate of removal of warfarin and trimethoprim obtained with the new catalyst was found twice the one observed by using TiO2 Degussa P25 and approximately 1.6 times faster for metoprolol, carbamazepine and gemfibrozil. An evaluation of the electrical energy per order magnitude of removal (EE/O) confirmed the enhanced performance of the new catalyst (24.3–31.8kWhm−3 rather than 32.8–39.3kWhm−3 for conventional TiO2) and that the performance is compound-dependent. Toxicity testing revealed that some assays are suitable for the investigation of bioactivity of treated waters containing contaminants of emerging concern at μgL−1 level. Specifically, the AMES Fluctuation Test, Fish Embryo Acute Toxicity Test and Green alga Selenastrum capricornutum test provided valuable results for an environmental impact assessment. On the other hand, the Daphnia magna and Vibrio fischeri acute toxicity tests were not sensitive enough to detect bioactivity in the samples analysed without prior pre-concentration.
Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay ...named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.
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•D-CAP identifies differentially bound chromatin proteins between different cell types•SMARCD1, identified by D-CAP, regulates ectodermal differentiation of ESCs•SMARCD1 is associated with bivalent genes and regulates H3K4me3/H3K27me3 distribution•SMARCD1 binds and regulates Klf4 directly and indirectly
Alajem et al. develop an assay that indicates differential association of SMARCD1 with chromatin in embryonic stem cells (ESCs) and early-differentiating cells. SMARCD1 is associated with bivalent genes in ESCs, regulates H3K4me3/H3K27me3 distribution, and binds and regulates the pluripotency factor Klf4.
Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells ...targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known.
In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state.
These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold.
This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Efficient photocatalytic activity in the visible range has been achieved with titanium oxide thin films. Doping, induced by oxygen deficiencies, is the key to the increased photodegradation ...properties of the film as both experimental and theoretical studies of the thin films demonstrate. The Figure shows an AFM image of the surface morphology of a TiO2 layer.
The 5â²-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational
analysis of the ODC IRES has led to the identification of sequences ...necessary for cap-independent translation of the ODC mRNA.
To discover novel IRES trans -acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC
mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type
ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular
IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements.
Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and
PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex.
Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide
insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.
Proteoforms expand genomic diversity and direct developmental processes. While high-resolution mass spectrometry has accelerated characterization of proteoforms, molecular techniques working to bind ...and disrupt the function of specific proteoforms have lagged behind. In this study, we worked to develop intrabodies capable of binding specific proteoforms. We employed a synthetic camelid nanobody library expressed in yeast to identify nanobody binders of different SARS-CoV-2 receptor binding domain (RBD) proteoforms. Importantly, employment of the positive and negative selection mechanisms inherent to the synthetic system allowed for amplification of nanobody-expressing yeast that bind to the original (Wuhan strain RBD) but not the E484 K (Beta variant) mutation. Nanobodies raised against specific RBD proteoforms were validated by yeast-2-hybrid analysis and sequence comparisons. These results provide a framework for development of nanobodies and intrabodies that target proteoforms.
•Pd nanoparticles were directly deposited onto Fecralloy foams by galvanic displacement.•Pd–Fecralloy foams were used as structured catalysts in oxidation reactions.•The oxidation of CO benefited ...from direct contact between Pd and Fecralloy substrate.•The oxidation of CH4 was not affected by low Pd dispersion.•Activity was good and Pd utilization was effective in both reactions.
The spontaneous deposition of Pd directly onto Fecralloy foam substrates was investigated as a simple and convenient approach to the preparation of structured technological catalysts for environmental applications, avoiding issues typically encountered with washcoated systems. Characterization of freshly prepared catalysts included ion chromatography and ICP–MS, to assess the Pd loading, cyclic voltammetry, to estimate the Pd surface area, SEM–EDX, XRD and TPR/TPO. Cyclic voltammetry, SEM–EDX and XRD were performed also on reaction aged samples. CO and CH4 catalytic oxidation under lean conditions were selected as test reactions occurring respectively in the low (100–300°C) and mid-high (300–600°C) temperature ranges. Pd–Fecralloy foam catalysts were found to guarantee good activity with an effective utilization of the noble metal content in spite of their relatively low Pd dispersion.
Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase ...separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.
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