Expression and purification of fluorinated proteins from mammalian suspension culture.

The site-specific encoding of noncanonical amino acids allows for the introduction of rationalized chemistry into a target protein. Of the methods that enable this technology, evolved tRNA and synthetase pairs offer the potential for expanded protein production and purification. Such an approach combines the versatility of solid-phase peptide synthesis with the scalable features of recombinant protein production. We describe the large scale production and purification of eukaryotic proteins bearing fluorinated phenylalanine in mammalian suspension cell preparations. Downstream applications of this approach include scalable recombinant protein preparation for ligand binding assays with small molecules and ligands, protein structure determination, and protein stability assays.

Efficacy of Boron Neutron Capture Therapy in Primary Central Nervous System Lymphoma: In Vitro and In Vivo Evaluation.

BACKGROUND: Boron neutron capture therapy (BNCT) is a nuclear reaction-based tumor cell-selective particle irradiation method. High-dose methotrexate and whole-brain radiation therapy (WBRT) are the recommended treatments for primary central nervous system lymphoma (PCNSL). This tumor responds well to initial treatment but relapses even after successful treatment, and the prognosis is poor as there is no safe and effective treatment for relapse. In this study, we aimed to conduct basic research to explore the possibility of using BNCT as a treatment for PCNSL. METHODS: The boron concentration in human lymphoma cells was measured. Subsequently, neutron irradiation experiments on lymphoma cells were conducted. A mouse central nervous system (CNS) lymphoma model was created to evaluate the biodistribution of boron after the administration of borono-phenylalanine as a capture agent. In the neutron irradiation study of a mouse PCNSL model, the therapeutic effect of BNCT on PCNSL was evaluated in terms of survival. RESULTS: The boron uptake capability of human lymphoma cells was sufficiently high both in vitro and in vivo. In the neutron irradiation study, the BNCT group showed a higher cell killing effect and prolonged survival compared with the control group. CONCLUSIONS: A new therapeutic approach for PCNSL is urgently required, and BNCT may be a promising treatment for PCNSL. The results of this study, including those of neutron irradiation, suggest success in the conduct of future clinical trials to explore the possibility of BNCT as a new treatment option for PCNSL.

Facile preparation of ethanediamine-ß-cyclodextrin modified capillary column for electrochromatographic enantioseparation of Dansyl amino acids.

Herein, the fabrication of a fascinating multifunctional cyclodextrin (CD) chiral stationary phase and its chiral separation performance in capillary electrochromatography are proposed. A facile interfacial polymerization was used to anchor ethanediamine-ß-cyclodextrin (EDA-ß-CD) polymerized with trimesoyl chloride (TMC) and to form the chiral stationary phase (CSP) composite onto the surface wall of the capillary. The characters of prepared columns were confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray Photoelectron Spectrometer (XPS), scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). This novel CSP offers multi-typical interactions including hydrogen bonding, π-interaction, hydrophobic and electrostatic interaction as well as steric effects which contribute to prominent chiral recognition for Dansyl-DL-amino acids in CEC modes. The EDA-ß-CD modified column showed eminent enantioseparation performance towards five Dansyl-DL-amino acids (the DL-forms of valine, threonine, leucine, phenylalanine, serine). Besides, the prepared columns were perfectly reproducible and stable. The relative standard deviations of the enantiomer retention times for intra-day (n = 5), inter-day (n = 3) runs and column-to-columns (n = 3) are below 0.54%, 1.35% and 4.89%, individually. This innovative chiral stationary phase shows a broader application view and scope in chiral recognition domain.

Application of achiral-chiral two-dimensional HPLC for separation of phenylalanine and tryptophan enantiomers in dietary supplement.

This study developed a two-dimensional heart-cutting LC method for the separation of amino acid enantiomers. Two approaches for achiral separation of amino acids, phenylalanine and tryptophan, were selected. Amino acids were separated on C18 or hydrophilic interaction liquid chromatography (HILIC) columns in first dimension after their enantiomer separation on a teicoplanin chiral column in second dimension. Mobile phases for both separation systems were optimized by testing different types of organic modifiers, concentrations of ion-pair agent (sodium 1-octanesulfonate), and ionic modifier (ammonium acetate). The resolution of enantiomers higher than 1.5 for both amino acids was achieved using a C18-teicoplanin coupled column separation system with mobile phases methanol/2 mM sodium 1-octanesulfonate (10:90 and 75:25, step gradient between achiral and chiral columns, respectively). The lower resolution of amino acid enantiomers (RS ˃ 0.9), but higher column efficiency, was achieved on a HILIC-teicoplanin separation system with mobile phases acetonitrile/50 mM ammonium acetate (90:10 and 80:20, step gradient between achiral and chiral columns, respectively). The developed heart-cutting 2D-LC methods were validated in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. The results suggested that the developed methods were applicable for the simultaneous determination of amino acid enantiomers in the dietary supplement. The sample contained l-phenylalanine and l-tryptophan.

Antimicrobial secondary metabolites of an endolichenic Aspergillus niger isolated from lichen thallus of Parmotrema ravum.

A new 6-benzyl-γ-pyrone (1), named aspergyllone was isolated from the culture filtrates of an endolichenic fungus Aspergillus niger Tiegh, obtained from lichen thallus Parmotrema ravum (Krog & Swinscow) Serus, collected in India. 1 was isolated for the first time from an endolichenic fungus together with six other known metabolites identified as aurasperones A (2) and D (3), asperpyrone A (4), fonsecinone A (5), carbonarone A (6) and pyrophen (7). The compounds were tested against a panel of human, plant, food borne and fish pathogens. Aspergyllone showed strong selective antifungal activity against Candida parapsilosis (Ashford) Langeron & Talice, with an IC50 of 52 µg/mL. Aurasperone A and pyrophen showed moderate to strong antimicrobial activity inhibiting seven different test pathogens, being pyrophen active with IC50 ranging from 35 to 97 µg/mL.[Formula: see text].

Waveguiding and SERS Simplified Raman Spectroscopy on Biological Samples.

Biomarkers detection at an ultra-low concentration in biofluids (blood, serum, saliva, etc.) is a key point for the early diagnosis success and the development of personalized therapies. However, it remains a challenge due to limiting factors like (i) the complexity of analyzed media, and (ii) the aspecificity detection and the poor sensitivity of the conventional methods. In addition, several applications require the integration of the primary sensors with other devices (microfluidic devices, capillaries, flasks, vials, etc.) where transducing the signal might be difficult, reducing performances and applicability. In the present work, we demonstrate a new class of optical biosensor we have developed integrating an optical waveguide (OWG) with specific plasmonic surfaces. Exploiting the plasmonic resonance, the devices give consistent results in surface enhanced Raman spectroscopy (SERS) for continuous and label-free detection of biological compounds. The OWG allows driving optical signals in the proximity of SERS surfaces (detection area) overcoming spatial constraints, in order to reach places previously optically inaccessible. A rutile prism couples the remote laser source to the OWG, while a Raman spectrometer collects the SERS far field scattering. The present biosensors were implemented by a simple fabrication process, which includes photolithography and nanofabrication. By using such devices, it was possible to detect cell metabolites like Phenylalanine (Phe), Adenosine 5-triphosphate sodium hydrate (ATP), Sodium Lactate, Human Interleukin 6 (IL6), and relate them to possible metabolic pathway variation.

Targeted expression and purification of fluorine labelled cold shock protein B by using an auxotrophic strategy.

High resolution NMR spectroscopy is a seminal method in modern structural biology to obtain insights into proteins' structure, dynamics and function at dilute condition as well as in a cell-like environment or even intracellularly. Usually, 1H, 15N or 13C nuclei are predominantly used for the characterization of the protein of interest. These measurements are limited due to the wealth of chemical shifts and background signals arising from all molecules present in the NMR test tube. On top of that, the protein under study has to be isotopically enriched in nitrogen and/or carbon nuclei enabling to overcome the inherently low natural abundance of 13C and 15N NMR active isotopes. In this way switching to 19F NMR spectroscopy strongly reduces the total amount of signals seen in an NMR spectrum as it turns off background signals and is for this reason extremely attractive for highly-resolved investigations of proteins performance measured directly in cells or in a cell-like environment. Here we show the effective expression and purification of cold shock protein B from Bacillus subtilis (BsCspB) using fluorine labelled phenylalanine or fluorine labelled tryptophan residues. We reveal that fluorine labelled BsCspB represents the same fold on a secondary as tertiary level as seen for the wild type protein independent of the labelling position illuminating the soft character of fluorine insertion. This experimental setup of targeted fluorine labelling sets a profound ground for a broad range of highly-resolved 19F NMR applications to be performed in a complex cellular environment.

Hydrophobic Amino Acid Content in Onions as Potential Fingerprints of Geographical Origin: The Case of Rossa da Inverno sel. Rojo Duro.

In this study, we were interested in comparing the amino acid profile in a specific variety of onion, Rossa da inverno sel. Rojo Duro, produced in two different Italian sites: the Cannara (Umbria region) and Imola (Emilia Romagna region) sites. Onions were cultivated in a comparable manner, mostly in terms of the mineral fertilization, seeding, and harvesting stages, as well as good weed control. Furthermore, in both regions, the plants were irrigated by the water sprinkler method and subjected to similar temperature and weather conditions. A further group of Cannara onions that were grown by micro-irrigation was also evaluated. After the extraction of the free amino acid mixture, an ion-pairing reversed-phase (IP-RP) HPLC method allowed for the separation and the evaporative light scattering detection of almost all the standard proteinogenic amino acids. However, only the peaks corresponding to leucine (Leu), phenylalanine (Phe), and tryptophan (Trp), were present in all the investigated samples and they were unaffected from the matrix interfering peaks. The use of the beeswarm/box plots revealed that the content of Leu and Phe were markedly influenced by the geographical origin of the onions (with *** p.

Electro-driven extraction of polar compounds using agarose gel as a new membrane: Determination of amino acids in fruit juice and human plasma samples.

In this work, polypropylene hollow fiber was replaced by agarose gel in conventional electro membrane extraction (EME) to develop a novel approach. The proposed EME method was then employed to extract two amino acids (tyrosine and phenylalanine) as model polar analytes, followed by HPLC-UV. The method showed acceptable results under optimized conditions. This green methodology outperformed conventional EME, and required neither organic solvents nor carriers. The effective parameters such as the pH values of the acceptor and the donor solutions, the thickness and pH of the gel, the extraction voltage, the stirring rate, and the extraction time were optimized. Under the optimized conditions (acceptor solution pH: 1.5; donor solution pH: 2.5; agarose gel thickness: 7mm; agarose gel pH: 1.5; stirring rate of the sample solution: 1000rpm; extraction potential: 40V; and extraction time: 15min), the limits of detection and quantification were 7.5ngmL-1 and 25ngmL-1, respectively. The extraction recoveries were between 56.6% and 85.0%, and the calibration curves were linear with correlation coefficients above 0.996 over a concentration range of 25.0-1000.0ngmL-1 for both amino acids. The intra- and inter-day precisions were in the range of 5.5-12.5%, and relative errors were smaller than 12.0%. Finally, the optimized method was successfully applied to preconcentrate, clean up, and quantify amino acids in watermelon and grapefruit juices as well as a plasma sample, and acceptable relative recoveries in the range of 53.9-84.0% were obtained.

Bacillus methylotrophicus ASWU-C2, a strain inhabiting hot desert soil, a new source for antibacterial bacillopyrone, pyrophen, and cyclopeptides.

A strain of Bacillus methylotrophicus was isolated from a soil sample collected in Aswan eastern desert, which is known for its extremely arid climate. After fermentation of the strain in liquid culture and subsequent extraction, a bioassay-guided isolation procedure yielded five compounds: 2-benzyl-4H-pyran-4-one, named bacillopyrone (1), pyrophen (2), macrolactin A (3) and the cyclopeptides malformin A1 (4), and bacillopeptin A (5). The structures were determined by interpretation of nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS) data. This is the first report on the isolation of compounds 1 and 2 from Bacillus species; compound 1 was reported previously as synthetic product. Bacillopyrone (1) exhibited moderate activity against the Gram-negative Chromobacterium violaceum with minimum inhibitory concentration 266.6 µg/mL, while macrolactin A (3) and malformin A1 (4) inhibited Staphylococcus aureus (minimum inhibitory concentrations 13.3 and 133.3 µg/mL, respectively).

Improving the extraction of l-phenylalanine by the use of ionic liquids as adjuvants in aqueous biphasic systems.

Polyethylene glycol (PEG) is widely used in the polymer-salt systems. However, the low polarity of the PEG-rich phase limits the application of aqueous biphasic systems (ABS). To overcome this disadvantage, a small quantity of ionic liquid (IL) was used as an adjuvant in ABS to enlarge the polarity range. Therefore, an innovative study involving addition of 4wt% imidazolium-based ILs to the PEG 600/NaH2PO4 ABS, aiming at controlling the phase behavior and extraction ability, was carried out. The phase diagrams, the tie-lines and the partitioning behavior of l-phenylalanine and ILs were studied in these systems. The results reveal that l-phenylalanine preferentially partitions for the PEG-rich phase. The addition of 4wt% IL to ABS controls the partitioning behavior of l-phenylalanine, which depends on the type of IL employed. Moreover, it is verified that increasing temperature lead to a decrease in the partition coefficient of l-phenylalanine.

[Separation of phenylalanine and methionine enantiomers by HPLC method: a comparison of stationary phase types]. / Separácia enantiomérov fenylalanínu a metionínu metódou HPLC: porovnanie typov stacionárnych fáz.

The paper deals with enantioselective separation of amino acids by the high performance liquid chromatography method. Separations of enantiomeric forms were tested on the chiral stationary phases based on ß-cyclodextrine, isopropyl carbamate cyclofructan 6, and the macrocyclic antibiotic teicoplanin. The best enantioseparation was obtained on the teicoplanin-based chiral stationary phase in the reversed-phase mode. UV spectrophotometric detection at 210 nm was used for detection of amino acids. The method was validated with respect to linearity, precision, limit of detection, limit of quantitation, and recovery. Limits of quantitation for phenylalanine and methionine enantiomers were 0.3 and 0.2 µg.ml-1, respectively. The HPLC method with teicoplanin-based chiral stationary phase was applied for analysis of dietary supplements.Key words: separation of enantiomers HPLC phenylalanine methionine.

Synthesis of CuPF6 -(S)-BINAP loaded resin and its enantioselectivity toward phenylalanine enantiomers.

A type of resin-anchored CuPF6 -(S)-BINAP was synthesized and identified. The PS-CuPF6 -(S)-BINAP resin was used to adsorb the phenylalanine enantiomers. The results showed that the adsorption capacity of PS-CuPF6 -(S)-BINAP resin toward L-phenylalanine was higher than that of resin toward D-phenylalanine. PS-CuPF6 -(S)-BINAP resin exhibited good enantioselectivity toward L-phenylalanine and D-phenylalanine. The influence of phenylalanine concentration, pH, adsorption time, and temperature on the enantioselectivity of the resin were investigated. The results showed that the enantioselectivity of the resin increased with increasing the phenylalanine concentration, pH, and adsorption time, while it decreased with an increase in temperature. The causes for these influences are discussed. The highest enantioselectivity (α = 2.81) was obtained when the condition of phenylalanine concentration was 0.05 mmol/mL, pH was 8, adsorption time was 12 h, and temperature 5°C. The desorption test for removing D/L-phenylalanine on PS-CuPF6 -(S)-BINAP resin was also investigated. The desorption ratios of D-phenylalanine and L-phenylalanine at pH of 1 were 95.7% and 94.3%, respectively. This result indicated that the PS-CuPF6 -(S)-BINAP resin could be regenerated by shaking with an acidic solution. The reusability of the PS-CuPF6 -(S)-BINAP resin was also assessed and the resin exhibited considerable reusability.

Facile and efficient electrochemical enantiomer recognition of phenylalanine using ß-Cyclodextrin immobilized on reduced graphene oxide.

This work demonstrates the facile and efficient preparation protocol of ß-Cyclodextrin-reduced graphene oxide modified glassy carbon electrode (ß-CD/RGO/GCE) sensor for an impressive chiral selectivity analysis for phenylalanine enantiomers. In this work, the immobilization of ß-CD over graphene sheets allows the excellent enantiomer recognition due to the large surface area and high conductivity of graphene sheets and extraordinary supramolecular (host-guest interaction) property of ß-CD. The proposed sensor was well characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and electrochemical impedance spectroscopy (EIS) techniques. The analytical studies demonstrated that the ß-CD/RGO/GCE exhibit superior chiral recognition toward L-phenylalanine as compared to D-phenylalanine. Under optimum conditions, the developed sensor displayed a good linear range from 0.4 to 40µM with the limit of detection (LOD) values of 0.10µM and 0.15µM for l- and D-phenylalanine, respectively. Furthermore, the proposed sensor exhibits good stability and regeneration capacity. Thus, the as-synthesized material can be exploited for electrochemical enantiomer recognition successfully.

Designing an Escherichia coli Strain for Phenylalanine Overproduction by Metabolic Engineering.

The phenylalanine pathway flux is controlled by two types of regulators, those that are specific to the pathway, as well as by global regulators. In order to demonstrate the importance of these global regulators, we first removed the pathway-specific regulators using all possible combinations of gene knockouts and knockins. We found that genes like aroG fbr performed best individually as well as in combination with other genes, while other genes like tyrA and tyrR worked only in combination with other modifications. Knocking in the tktA gene under a tyrR promoter and knocking out pykF further increased phenylalanine production demonstrating that the supply of precursor via PEP and E4P is also a rate-limiting step. Finally, we tested the role of global regulators on this deregulated pathway and found that Fis overexpression helps in both enhancing and sustaining the flux through this pathway. This work opens up the possibility of using global regulators in synergy with pathway-specific modifications to enhance product yields.

Three new amino acid derivatives from edible mushroom Pleurotus ostreatus.

Three new amino acid derivatives, oxalamido-L-phenylalanine methyl ester (1), oxalamido-L-leucine methyl ester (2), and lumichrome hydrolyzate (3), together with nine known compounds (4-12), were isolated from the solid culture of edible mushroom Pleurotus ostreatus. Their structures were elucidated on the basis of extensive spectroscopic analysis. The absolute configurations of 1 and 2 were established by the chiral synthesis and confirmed by circular dichroism (CD) analysis of their total synthesis products and natural isolates. All new compounds were evaluated for their antioxidant effects, antimicrobial activities, and cytotoxic activity. Compounds 1-3 showed weak antifungal activities against Candida albicans with minimum inhibitory concentration (MIC) value of 500 µg/ml.

High resolution of racemic phenylalanine with dication imidazolium-based chiral ionic liquids in a solid-liquid two-phase system.

A novel solid-liquid two-phase system was developed for the chiral separation of racemic phenylalanine with new dication imidazolium-based chiral ionic liquids. Preliminary experiments showed distinct enantioselectivity in amino acid extraction with the novel solid-liquid two-phase system, more L-enantiomer of amino acid cooperatively interacted with ionic liquids and copper ions to be the solid phase. Various factors, including the alkyl chain length of cations of ionic liquids, the amount of copper acetate, the ratio of n(ILs)/n(Cu2+), the amount of water and racemic phenylalanine, the resolution time together with the resolution temperature, were systematically investigated for their influence on resolution efficiency. The results showed that, under a certain condition, the enantiomeric excess value and the yield of phenylalanine in liquid phase (mainly containing D-enantiomer) were 67.8% and 96.5%, the enantiomeric excess value and the yield of phenylalanine in solid phase (mainly containing L-enantiomer) were 99.2% and 85.2%. Finally, 2D NMR technology, infrared spectroscopy and molecular simulation method were used to study the interaction mechanism. The results indicated that L-enantiomer of phenylalanine interacts more strongly with chiral ILs and Cu2+. The novel system has characteristics of free-organic solvent, simple operation, fast separation process and very high resolution efficiency for racemic phenylalanine. This work could provide a new and alternative resolution approach for other chiral separations.

Isoacceptor specific characterization of tRNA aminoacylation and misacylation in vivo.

Amino acid misincorporation during protein synthesis occurs due to misacylation of tRNAs or defects in decoding at the ribosome. While misincorporation of amino acids has been observed in a variety of contexts, less work has been done to directly assess the extent to which specific tRNAs are misacylated in vivo, and the identity of the misacylated amino acid moiety. Here we describe tRNA isoacceptor specific aminoacylation profiling (ISAP), a method to identify and quantify the amino acids attached to a tRNA species in vivo. ISAP allows compilation of aminoacylation profiles for specific isoacceptors tRNAs. To demonstrate the efficacy and broad applicability of ISAP, tRNAPhe and tRNATyr species were isolated from total aminoacyl-tRNA extracted from both yeast and Escherichia coli. Isolated aminoacyl-tRNAs were washed until free of detectable unbound amino acid and subsequently deacylated. Free amino acids from the deacylated fraction were then identified and quantified by mass spectrometry. Using ISAP allowed quantification of the effects of quality control on the accumulation of misacylated tRNA species under different growth conditions.

Preparation of imprinted cryogel cartridge for chiral separation of l-phenylalanine.

l-Phe-imprinted cryogel cartridge was prepared for the chiral separation of l-Phe. N-Methacryloyl l-phenylalanine (MAPA) was used as a functional monomer for complexing with l-Phe. The selectivity of the membranes was investigated by using d-Phe, l-Trp, and d-Trp as competitor molecules. The PHEMAPA-l-Trp membranes were 6.4, 4.3, and 5.5 times more selective for l-Phe than d-Phe, l-Trp, and d-Trp, respectively. The PHEMAPA-l-Phe cryogel cartridge was incorporated into the fast protein liquid chromatography (FPLC) equipment and was able to separate D,l-Phe racemic mixture efficiently. The PHEMAPA-l-Phe membranes were shown to be reusable many times without significant loss of the adsorption capacity.

Simultaneous and selective decarboxylation of L-serine and deamination of L-phenylalanine in an amino acid mixture--a means of separating amino acids for synthesizing biobased chemicals.

Amino acids (AAs) obtained from the hydrolysis of biomass-derived proteins are interesting feedstocks for the chemical industry. They can be prepared from the byproduct of biofuel production and agricultural wastes. They are rich in functionalities needed in petrochemicals, providing the opportunity to save energy, reagents, and process steps. However, their separation is required before they can be applied for further applications. Electrodialysis (ED) is a promising separation method, but its efficiency needs to be improved when separating AAs with similar isoelectric points. Thus, specific conversions are required to form product with different charges. Here we studied the enzymatic conversions which can be used as a means to aid the ED separation of neutral AAs. A model mixture containing L-serine, L-phenylalanine and L-methionine was used. The reactions of L-serine decarboxylase and L-phenylalanine ammonia-lyase were employed to specifically convert serine and phenylalanine into ethanolamine and trans-cinnamic acid. At the isoelectric point of methionine (pH 5.74), the charge of ethanolamine and trans-cinnamic acid are +1 and -1, therefore facilitating potential separation into three different streams by electrodialysis. Here the enzyme kinetics, specificity, inhibition and the operational stabilities were studied, showing that both enzymes can be applied simultaneously to aid the ED separation of neutral AAs.