Chromatographic Purification Procedure Research Articles

Isolation by multistep chromatography improves the consistency of secreted recombinant influenza neuraminidase antigens

Recombinant protein-based approaches are ideally suited for producing vaccine antigens that are not overly abundant in viruses, such as influenza neuraminidase (NA). However, obtaining sufficient quantities of recombinant viral surface antigens remains challenging, often resulting in the use of chimeric proteins with affinity tags that can invariably impact the antigen’s properties. Here, we developed multistep chromatography approaches for purifying secreted recombinant NA (rNA) antigens that are derived from recent H1N1 and H3N2 viruses and produced using insect cells. Analytical analyses showed that these isolation procedures yielded homogenous tetrameric rNA preparations with consistent specific activities that were not possible from a common immobilized metal affinity chromatography purification procedure. The use of classical chromatography improved the rNA tetramer homogeneity by removing the requirement of the N-terminal poly-histidine affinity tag that was shown to promote higher order rNA oligomer formation. In addition, these procedures reduced the specific activity variation by eliminating the exposure to Ni2+ ions and imidazole, with the latter showing pH and NA subtype dependent effects. Together, these results demonstrate that purification by multistep chromatography improves the homogeneity of secreted rNAs and eliminates the need for affinity tag-based approaches that can potentially alter the properties of these recombinant antigens.

Tf2O as a CF3 Source for the Synthesis of Trifluoromethoxylation Reagent nC4F9SO3CF3.

Described herein is the convenient synthesis of an efficient trifluoromethoxylation reagent, nC4F9SO3CF3, by using cheap and widely available reagents and without the need of any tedious column chromatography purification procedure.

A Simple Micropreparative Gel Electrophoresis Technique for Purification of Nanoscale Materials

Many biochemical, biomedical, and material applications hinge on the ability to effectively separate and purify nanoscale materials. Though this need is largely addressed with biological macromolecules using a variety of chromatographic and electrophoretic purification techniques, such techniques are usually laborious, time-consuming, and often require complex and costly instalments that are inaccessible to most laboratories. Synthetic nanoparticles face similar purification challenges, often relying on techniques that are material-specific. In this work, we introduce a versatile micro-preparative (MP) method based on polyacrylamide gel electrophoresis (PAGE) to purify biological samples containing proteins, nucleic acids, and complex bioconjugates, as well as synthetic nanoparticles based on graphene quantum dots (GQDs). Using a conventional vertical slab PAGE, we demonstrate the extraction of purified DNA, proteins, and DNA-protein bioconjugates from their respective mixtures using MP-PAGE. We apply this system to recover DNA from a ladder mixture with yields of up to 90%, compared to the 58% yield obtained using specialized commercial devices. We also demonstrate the purification of folded enhanced yellow fluorescence protein (EYFP) from crude cell extract with 90% purity, comparable to purities achieved using a two-step size exclusion and immobilized metal-ion affinity chromatography purification procedure. Moreover, we demonstrate the successful isolation of an EYFP-DNA bioconjugate that otherwise could not be processed using the two-step chromatography procedure. Finally, the technique was further extended to demonstrate size-dependent separation of a commercial mixture of GQDs into three different fractions with distinct optical properties. MP-PAGE thus offers a rapid and versatile means of purifying biological and synthetic nanomaterials without the need for specialized equipment.

Precise measurements of δ88/86Sr for twenty geological reference materials by double-spike MC-ICP-MS

Analytical method using double-spike and MC-ICP-MS can provide high efficiency of measurement for stable Sr isotopic compositions (δ88/86Sr). Here we report a method for high precision measurement of δ88/86Sr using MC-ICP-MS. A84Sr–87Sr double-spike was used to correct instrumental mass bias of stable Sr isotopes. Strontium was purified by a cation exchange resin (AG50W-X12, 200–400 mesh), and for samples with high Er, Yb, Lu, and Hf contents, an additional chromatographic purification procedure was applied. The potential effects of doubly charged ion isobaric inferences and different measurement conditions were rigorously evaluated to improve the analytical precision. Data quality was monitored by measuring three USGS reference materials (BHVO-2, AGV-2, and G-2) and two in-house reference materials, giving the long-term precision of 0.03‰ (2SD) for δ88/86Sr. Twenty reference materials, including nine igneous rocks, four sedimentary rocks, two sediments, and five soils, have δ88/86Sr values range from −0.06‰ to 0.35‰, showing resolvable isotope fractionation during magmatic and supergene processes. This study provides a useful standard dataset for inter-laboratory comparison of stable Sr isotope studies.

Production of Structured RNA Fragments by In Vitro Transcription and HPLC Purification.

The understanding of the functional importance of RNA has increased enormously in the last decades. This has required research on the RNA molecules themselves, with the concomitant need for obtaining purified RNA samples, such as for structural studies by NMR or other methods. The main method to create labeled and unlabeled RNA, T7 in vitro transcription, suffers from sequence-dependent yield and often low homogeneity for short constructs (<100 nt) and requires laborious purification. Additionally, the design of structured RNA fragments mimicking the structure of a larger biological RNA is often not straightforward. Secondary structure simulations can be used to make reliable predictions about the folding of a particular RNA fragment. In this article, we describe how to design an RNA construct of interest from a larger sequence, and we combine several previously published improvements of the in vitro transcription method, such as the use of 2'-methoxy modifications and dimethyl sulfoxide or the use of tandem repeats, to increase yield and purity of in vitro-transcribed RNA. Together with a high-performance liquid chromatography (HPLC) purification procedure using both reversed-phase ion-pairing and ion-exchange HPLC, we provide a robust protocol to obtain highly pure RNA of short to intermediate length in large quantities. The protocol optimizes yield, especially for RNA starting with nucleotides other than G. At the same time, it is simplified, and the required time is reduced. The protocols described here constitute a versatile pipeline for the production of purified RNA samples and are suitable for users with little experience in liquid chromatography. © 2021 The Authors. Basic Protocol 1: RNA construct design Basic Protocol 2: DNA template production and in vitro transcription Alternate Protocol: Tandem transcription and RNase H cleavage Basic Protocol 3: Reversed-phase ion-pairing HPLC purification Basic Protocol 4: Ion-exchange HPLC purification.

Expression and purification of the human tumor suppressor protein RNASET2 in CHO–S cells

Members of the T2 extracellular ribonucleases family have long been reported as stress response proteins, often involved in host defence, in many different taxonomic groups. In particular, the human RNASET2 protein (hRNASET2) has been reported as an extracellular tumor suppressor protein, endowed with the ability to act as an “alarmin” signalling molecule following its expression and secretion in the tumor microenvironment by cancer cells and the subsequent recruitment and activation of cells belonging to the host innate immune system. Many in vitro and in vivo assays have been recently reported in support of the oncosuppressive role of hRNASET2: most of them relied on genetically engineered cell lines and the use of recombinant proteins from non-mammalian sources. In order to ensure a human-like glycosylation pattern, here we report for the first time the expression of recombinant hRNASET2 in the CHO–S cell line. We established a simple one-step chromatographic purification procedure that resulted in the production of 5 mg of endotoxin-free hRNASET2 per liter of culture, with a >95% purity degree. hRNASET2 expressed in CHO–S cells displayed a high degree of glycosylation homogeneity and a secondary structure content in agreement with that determined from the crystal structure. Indeed, recombinant hRNASET2 was active at both enzymatic and functional level, as stated by a biological activity assay. The availability of a pure, homogeneous recombinant human RNASET2 would provide a key tool to better investigate its non cell-autonomous roles in the context of cancer development and growth.

A TLC-HPLC Method for Determination of Thiamphenicol in Pig, Chicken, and Fish Feedstuffs

An effective thin-layer chromatography (TLC) purification procedure coupled to high-performance liquid chromatography (HPLC) method was developed for the determination of thiamphenicol (TAP) in pig, chicken, and fish feedstuffs. The feedstuff samples were extracted with ethyl acetate, defatted with n-hexane saturated with acetonitrile, and further purified by TLC. The chromatographic separation was performed on a Waters Symmetry C18 column using an isocratic procedure with acetonitrile-water (21.7:78.3, v/v) at 0.6 mL/min. The ultraviolet (UV) detector was set at a wavelength of 225 nm. The TAP concentrations in feedstuff samples were quantified using a standard curve. Good linear correlations (y = 162,630x − 2381.7, r > 0.9998) were achieved within the concentration range of 0.05–10.00 μg/mL. The recoveries of TAP spiked at levels of 1, 10, and 100 μg/g ranged from 82.0 to 114.9% with the intra-day and inter-day relative standard deviation (RSD) less than 9.0%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.03 mg/kg for pig feedstuffs, 0.02 and 0.04 mg/kg for chicken feedstuffs, and 0.02 and 0.03 mg/kg for fish feedstuffs, respectively. This reliable, simple, and cost-effective method could be applied to the routine monitoring of TAP in animal feedstuffs.

An Organocatalytic Novel Synthesis of Polyfunctionalized Bis-2,5-dihydrofuran-3-carboxylates via Domino-MCR Strategy

For the first time, an elegant, metal-free and concise route using a pseudo five-component reaction of dimethyl acetylenedicarboxylate, mono/bis- aromatic aldehydes and amines with trifluoroacetic acid as an effective organocatalyst to afford novel and diverse substituted bis-2,5-dihydrofuran-3-carboxylates in good yields at ambient temperature has been described. The prominent features of this protocol are the energy sustainability, wide range of functional group tolerance, shorter reaction time, no column chromatographic purification, facile experimental and work-up procedure. The present one-step, cascade synthetic approach is straightforward that could avoid hazardous reagents/solvents and is turned to be a benign chemistry for the preparation of potent bis-O-heterocyclics on a multigram scale.

Development of a high-performance liquid chromatography method for the determination of florfenicol in animal feedstuffs.

An effective thin layer chromatography (TLC) purification procedure coupled to high-performance liquid chromatography (HPLC) method was developed for the determination of florfenicol (FF) in pig, chicken and fish feedstuffs. The feedstuff samples were extracted with ethyl acetate, defatted with n-hexane saturated with acetonitrile, and further purified by TLC. The chromatographic separation was performed on a Waters Symmetry C18 column using an isocratic procedure with acetonitrile-water (35:65, v/v) at 0.6mL/min. The ultraviolet (UV) detector was set at a wavelength of 225nm. The FF concentrations in feedstuff samples were quantified using a standard curve. Good linear correlations (y=159075x-15054, r>0.9999) were achieved within the concentration range of 0.05-200μg/mL. The recoveries of FF spiked at levels of 1, 100 and 1000μg/g ranged from 80.6% to 105.3% with the intra-day and inter-day relative standard deviation (RSD) less than 9.3%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.06mg/kg for pig feedstuffs, 0.02 and 0.07mg/kg for chicken feedstuffs, and 0.02 and 0.05mg/kg for fish feedstuffs, respectively. This reliable, simple and cost-effective method could be applied to the routine monitoring of FF in animal feedstuffs.

A clean and simple method for deprotection of phosphines from borane complexes

Abstract Simple, efficient, clean, and stereospecific protocols of protection of phosphorus atom with borane and deprotection from the borane complexes of the tertiary phosphines in mild conditions are reported. The proposed protection/deprotection reactions tolerate a range of functional groups and lead to pure products with excellent yield with no need for application of chromatographic or crystallisation purification procedures. For the first time mechanisms of the reactions of phosphine protection and deprotection have been studied based on experimental kinetic data as well as quantumchemical calculations, which allows designing reaction conditions suitable for a given substrate.

Purification of HIV-1 gag virus-like particles and separation of other extracellular particles

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100⿿200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1ÿ109 particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

Process Development of the Synthesis and Purification of a Reactive Immuno-PET Conjugate Intermediate

We describe a practical multigram scale synthesis of the reactive immuno-PET linker-chelator GNE-605 from the commercially available starting material desferrioxamine B mesylate. A modified three-step synthetic procedure was developed that significantly reduces the amount of solvents and reagents required as compared to previous literature. Additionally, due to the unique handling challenges posed by this reactive intermediate, a liquid chromatography purification and lyophilization procedure was developed to allow for the first isolation of GNE-605 as a solid material.

Expression, Purification and Crystallization of Recombinant Arabidopsis Monogalactosyldiacylglycerol Synthase (MGD1)

In plant cells, galactolipids are predominant, representing up to 50% of the lipid content in photosynthetic tissues. Galactolipid synthesis is initiated by MGDG synthases (MGDs), which use UDP-galactose as a donor sugar and diacylglycerol (DAG) as acceptor, to form monogalactosyldiacylglycerol (MGDG). This protocol is used to produce a recombinant form of Arabidopsis thaliana (A. thaliana) monogalactosyldiacylglycerol synthase 1 (MGD1) protein, in Escherichia coli (E. coli), using a two-step chromatographic purification procedure. The protein is easily expressed and purified to milligram quantities, suitable for biochemical and structural studies. The crystallization of MGD1 is also described.

Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography.

Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impact on swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccine candidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizes were examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. Q Sepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some host cell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6B and CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPs after chromatography on Heparin Sepharose CL-6B was only 4-7% and the recovery of VLPs was 5-7%. Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purity of about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 monolith was 15-18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electron microscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate was developed using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith.

Deuterated nucleotides as chemical probes of RNA structure: a detailed protocol for the enzymatic synthesis of a complete set of nucleotides specifically deuterated at ribose carbons

Abstract We describe here a detailed protocol for the synthesis of ribonucleotides specifically deuterated at each ribose carbon atom. We synthesized 20 specifically deuterated ribonucleotides: ATP, CTP, GTP, and UTP, each of which contained one of five deuterated riboses (either 1′-D, 2″-D, 3′-D, 4′-D, or 5′,5″-D2). Our synthetic approach is inspired by the pioneering work of Tolbert and Williamson, who developed a method for the convenient one-pot enzymatic synthesis of nucleotides (Tolbert, T. J. and Williamson, J. R. (1996) J. Am. Chem. Soc. 118, 7929–7940). Our protocol consists of a comprehensive list of required chemical and enzymatic reagents and equipment, detailed procedures for enzymatic assays and nucleotide synthesis, and chromatographic procedures for purification of deuterated nucleotides. As an example of the utility of specifically deuterated nucleotides, we used them to synthesize specifically deuterated sarcin/ricin loop (SRL) RNA and measured the deuterium kinetic isotope effect on hydroxyl radical cleavage of the SRL.

돌외 잎 추출물의 콜라겐 합성 증진 성분 규명

In order to discover ingredients for wrinkle-care cosmetics, we prepared 70% ethanol extract from Gynostemma pentaphyllum and examined its activity on collagen synthesis using fibroblast HDFn cells. The G. pentaphyllum extract induced the production of type I procollagen in a dose-dependent manner without showing cell toxicity. The active constituents were isolated from the extract by solvent fractionation and chromatographic purification procedures. NMR data and literature studies led to determine the two isolated compounds as the flavonoid glycosides such as ombuine 3-O-rutinoside (1) and quercetin 3-O-rutinoside (2). The activity screening tests showed that the isolates 1 and 2 induced the production of type I procollagen in a dose-dependent manner. These results suggested that G. pentaphyllum extract containing the flavonoids 1 and 2 could be useful as an active ingredient for wrinkle-care cosmetics.

Automated analytical standard production with supercritical fluid chromatography for the quantification of bioactive C17-polyacetylenes: A case study on food processing waste

Food processing enterprises produce enormous amounts of organic waste that contains valuable phytochemicals (e.g. C17-polyacetylenes). Knowledge on the phytochemicals content is a first step towards valorisation. Quantification of C17-polyacetylenes is however often hampered by the lack of commercially available standards or by tedious multistep in-house standard production procedures. In the current study, a new and straightforward supercritical fluid chromatography purification procedure is described for the simultaneous production of 2 analytical C17-polyacetylene standards. Respectively, 5 and 6mg of falcarinol and falcarindiol were purified in 17h on analytical scale. After confirming the identity and quality (97% purity) by Nuclear Magnetic Resonance, accurate mass-Mass Spectrometry (am-MS) and Photo Diode Array (PDA) detection the C17-polyacetylene standards were used for the analysis of industrial vegetable waste with Liquid Chromatography coupled to PDA and am-MS detection. Measurements showed varying concentrations of C17-polyacetylenes in the organic waste depending on its nature and origin.

Human mitochondrial NDUFS3 protein bearing Leigh syndrome mutation is more prone to aggregation than its wild-type

NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCl induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly.

Expanding the structural repertoire of β/α Ant-Pro (anthranilic acid-proline) oligomers into γ/α 2-Amb-Pro (2-aminomethyl benzoic acid-proline) oligomers

In this article, we report a novel class of heterogeneous synthetic oligomers featuring the conformationally constrained amino acid residues – 2-aminomethyl benzoic acid (2-Amb) and proline (Pro) in repeating sequences. Oligomers as large as hexadecamers featuring the conformationally restricted γ/α 2-Amb-Pro motif have been assembled using solution-phase Boc strategy, following multi-step synthetic sequences starting from the commercially available O-toluic acid. EDC-mediated peptide coupling has been found to be optimum for the assembly of the relatively non-polar oligomers, which could be readily purified by the standard column chromatographic purification procedures. This study offers considerable prospects of expanding the structural repertoire of β/α Ant-Pro motif, which has been described earlier to assume right-handed helical architecture displaying robust nine-membered-ring closed network of hydrogen-bonding interactions, into γ/α 2-Amb-Pro motif.

Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip

Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.