SECRET AGENT O-GlcNAcylates Hundreds of Proteins Involved in Diverse Cellular Processes in Arabidopsis.

O-GlcNAcylation is a critical post-translational modification of proteins observed in both plants and animals and plays a key role in growth and development. While considerable knowledge exists about over 3000 substrates in animals, our understanding of this modification in plants remains limited. Unlike animals, plants possess two putative homologs: SECRET AGENT (SEC) and SPINDLY, with SPINDLY also exhibiting O-fucosylation activity. To investigate the role of SEC as a major O-GlcNAc transferase in plants, we utilized lectin-weak affinity chromatography enrichment and stable isotope labeling in Arabidopsis labeling, quantifying at both MS1 and MS2 levels. Our findings reveal a significant reduction in O-GlcNAc levels in the sec mutant, indicating the critical role of SEC in mediating O-GlcNAcylation. Through a comprehensive approach, combining higher-energy collision dissociation and electron-transfer high-energy collision dissociation fragmentation with substantial fractionations, we expanded our GlcNAc profiling, identifying 436 O-GlcNAc targets, including 227 new targets. The targets span diverse cellular processes, suggesting broad regulatory functions of O-GlcNAcylation. The expanded targets also enabled exploration of crosstalk between O-GlcNAcylation and O-fucosylation. We also examined electron-transfer high-energy collision dissociation fragmentation for site assignment. This report advances our understanding of O-GlcNAcylation in plants, facilitating further research in this field.

A rapid, tag-free way to purify functional GPCRs.

G protein-coupled receptors (GPCRs) play diverse signaling roles and represent major pharmaceutical targets. Consequently, they are the focus of intense study, and numerous advances have been made in their handling and analysis. However, a universal way to purify GPCRs has remained elusive, in part because of their inherent instability when isolated from cells. To address this, we have developed a general, rapid, and tag-free way to purify GPCRs. The method uses short peptide analogs of the Gα subunit C terminus (Gα-CT) that are attached to chromatography beads (Gα-CT resin). Because the Gα-CT peptides bind active GPCRs with high affinity, the Gα-CT resin selectively purifies only active functional receptors. We use this method to purify both rhodopsin and the ß2-adrenergic receptor and show they can be purified in either active conformations or inactive conformations, simply by varying elution conditions. While simple in concept-leveraging the conserved GPCR-Gα-CT binding interaction for the purpose of GPCR purification-we think this approach holds excellent potential to isolate functional receptors for a myriad of uses, from structural biology to proteomics.

Site-Specific Activity-Based Protein Profiling Using Phosphonate Handles.

Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography-enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID-ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID-ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID-ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID-ABPP providing the advantage of direct activity-based probe interaction site identification.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands.

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1 × 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80 nM, a Tm of >70 °C, and a t1/2 in 0.1 mg/ml trypsin of >20 h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10 min of cleaning-in-place with 0.1 M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.

Targeting of bone morphogenetic protein complexes to heparin/heparan sulfate glycosaminoglycans in bioactive conformation.

Bone morphogenetic proteins (BMP) are powerful regulators of cellular processes such as proliferation, differentiation, and apoptosis. However, the specific molecular requirements controlling the bioavailability of BMPs in the extracellular matrix (ECM) are not yet fully understood. Our previous work showed that BMPs are targeted to the ECM as growth factor-prodomain (GF-PD) complexes (CPLXs) via specific interactions of their PDs. We showed that BMP-7 PD binding to the extracellular microfibril component fibrillin-1 renders the CPLXs from an open, bioactive V-shape into a closed, latent ring shape. Here, we show that specific PD interactions with heparin/heparan sulfate glycosaminoglycans (GAGs) allow to target and spatially concentrate BMP-7 and BMP-9 CPLXs in bioactive V-shape conformation. However, targeting to GAGs may be BMP specific, since BMP-10 GF and CPLX do not interact with heparin. Bioactivity assays on solid phase in combination with interaction studies showed that the BMP-7 PD protects the BMP-7 GF from inactivation by heparin. By using transmission electron microscopy, molecular docking, and site-directed mutagenesis, we determined the BMP-7 PD-binding site for heparin. Further, fine-mapping of the fibrillin-1-binding site within the BMP-7 PD and molecular modeling showed that both binding sites are mutually exclusive in the open V- versus closed ring-shape conformation. Together, our data suggest that targeting exquisite BMP PD-binding sites by extracellular protein and GAG scaffolds integrates BMP GF bioavailability in a contextual manner in development, postnatal life, and connective tissue disease.

Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles.

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

Continuous multi-column capture of monoclonal antibodies with convective diffusive membrane adsorbers.

Downstream processing is the bottleneck in the continuous manufacturing of monoclonal antibodies (mAbs). To overcome throughput limitations, two different continuous processes with a novel convective diffusive protein A membrane adsorber (MA) were investigated: the rapid cycling parallel multi-column chromatography (RC-PMCC) process and the rapid cycling simulated moving bed (RC-BioSMB) process. First, breakthrough curve experiments were performed to investigate the influence of the flow rate on the mAb dynamic binding capacity and to calculate the duration of the loading steps. In addition, customized control software was developed for an automated MA exchange in case of pressure increase due to membrane fouling to enable robust, uninterrupted, and continuous processing. Both processes were performed for 4 days with 0.61 g L-1 mAb-containing filtrate and process performance, product purity, productivity, and buffer consumption were compared. The mAb was recovered with a yield of approximately 90% and productivities of 1010 g L-1 d-1 (RC-PMCC) and 574 g L-1 d-1 (RC-BioSMB). At the same time, high removal of process-related impurities was achieved with both processes, whereas the buffer consumption was lower for the RC-BioSMB process. Finally, the attainable productivity for perfusion bioreactors of different sizes with suitable MA sizes was calculated to demonstrate the potential to operate both processes on a manufacturing scale with bioreactor volumes of up to 2000 L.

Soluble expression of recombinant human interleukin-2 in Escherichia coli and its facile production.

Recombinant human interleukin-2 (rhIL-2) represents one of the most difficult-to-produce cytokines in E. coli due to its extreme hydrophobicity and high tendency to formation of inclusion bodies. Refolding of rhIL-2 inclusion bodies always represents cumbersome downstream processes and low production efficiency. Herein, we disclosed a fusion strategy for efficiently soluble expression and facile production of rhIL-2 in E. coli Origami B (DE3) host. A two-tandem SUMO fusion partner (His-2SUMO) with a unique SUMO protease cleavage site at C-terminus was devised to fuse with the N-terminus of rhIL-2 and the fusion protein (His-2SUMO-rhIL-2) was almost completely expressed in a soluble from. The fusion partner could be efficiently removed by Ulp1 cleavage and the rhIL-2 was simply produced by a two-step Ni-NTA affinity chromatography with a considerable purity and whole recovery. The eventually obtained rhIL-2 was well-characterized and the results showed that the purified rhIL-2 exhibits a compact and ordered structure. Although the finally obtained rhIL-2 exists in a soluble aggregates form and the aggregation probably has been occurred during expression stage, the soluble rhIL-2 aggregates remain exhibit comparable bioactivity with the commercially available rhIL-2 drug formulation.

Purification of thioredoxin reductase from Spirulina platensis by affinity chromatography and investigation of kinetic properties.

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the TrxR enzyme was purified for the first time from Spirulina platensis, an algae the most grown and also used as a nutritional supplement in the world. A two-step purification process was used: preparation of the homogenate and 2',5'-ADP sepharose 4B affinity chromatography. The enzyme was purified with a purification fold of 1059.51, a recovery yield of 9.7 %, and a specific activity of 5.77 U/mg protein. The purified TrxR was tested for purity by SDS-PAGE. The molecular weight of its subunit was found to be about 45 kDa. Optimum pH, temperature and ionic strength of the enzyme were pH 7.0, 40 °C and 750 mM in phosphate buffer respectively. The Michaelis constant (Km) and maximum velocity of enzyme (Vmax) values for NADPH and 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) are 5 µM and 2.2 mM, and 0.0033 U/mL and 0.0044 U/mL, respectively. Storage stability of the purified enzyme was determined at several temperatures. The inhibition effects of Ag+, Cu2+, Al3+ and Se4+ metal ions on the purified TrxR activity were investigated in vitro. While Se4+ ion increased the enzyme activity, other tested metal ions showed different type of inhibitory effects on the Lineweaver-Burk graphs.

Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification.

The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads for His-tagged protein purification. We investigated the binding of His-tagged Mpro on the Ni-M/A beads, referred to as Ni-M/A-Mpro, and assessed the elution efficiency of Mpro from the beads. Our examination involved FTIR, EDS, XRD, SDS-PAGE, and Western blotting methods. FTIR spectra revealed notable changes in the stretching patterns and intensities of hydroxyl, amine, carbonyl, imine and amide chemical groups, when Mpro protein was present in the Ni-M/A sample. XRD spectra demonstrated the occurrence of two Nickel peaks at 35-40 deg and 40-45 deg in Ni-M/A, but only one nickel peak at 35-40 deg in Ni-M/A-Mpro, indicating the binding of Mpro on the Nickel ions. EDS analysis reported a decrease in the concentration of Nickel on the surface of Ni-M/A from 16% to 7% when Mpro protein was loaded into the stationary phase. Importantly, our data indicated that the purity of the His-tagged protein Mpro after purification reached 97% after just one-step purification using the Ni-M/A stationary phase. Moreover, the binding capacity of Ni-M/A for Mpro was approximately 5.2 mg/g with recovery efficiency of 40%. Our results suggested Ni-M/A as a highly potential solid phase for affinity chromatography in the purification of His-tagged protein.

Affinity gel synthesis from the p-aminobenzoic acid derivative 4-amino-2-methylbenzoic acid and purification of polyphenol oxidase from various plant sources.

The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (Ki: 0.07 ± 0.03 µM), mushroom (Ki: 0.7 ± 0.3 µM), and eggplant (Ki: 4.8 ± 1.2 µM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (Ki: 1112 ± 324 µM), mushroom (Ki: 567 ± 81 µM), and eggplant (Ki: 2016.7 ± 805.6 µM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO.

Scalable dual column cation exchange affinity chromatography based platform process for recombinant protein purification.

A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting as an initial affinity tag. Combining the heparin-binding tag (HB-tag) with the self-removing iCapTag™ provides a means for removing both tags in a subsequent self-cleaving step. The result is a convenient platform for the purification of diverse tagless proteins with a range of isoelectric points and molecular weights. In this work, we demonstrate a dual column process in which the tagged protein of interest is first captured from an E. coli cell lysate using a cation exchange column via a fused heparin-binding affinity tag. The partially purified protein is then diluted and loaded onto an iCapTag™ split-intein column, washed, and then incubated overnight to release the tagless target protein from the bound tag. Case studies are provided for enhanced green fluorescent protein (eGFP), beta galactosidase (ßgal), maltose binding protein (MBP) and beta lactamase (ßlac), where overall purity and host cell DNA clearance is provided. Overall, the proposed dual column process is shown to be a scalable platform technology capable of accessing both the high dynamic binding capacity of ion exchange resins and the high selectivity of affinity tags for the purification of recombinant proteins.

Target fishing reveals PfPYK-1 and PfRab6 as potential targets of an antiplasmodial 4-anilino-2-trichloromethylquinazoline hit compound.

We present investigations about the mechanism of action of a previously reported 4-anilino-2-trichloromethylquinazoline antiplasmodial hit-compound (Hit A), which did not share a common mechanism of action with established commercial antimalarials and presented a stage-specific effect on the erythrocytic cycle of P. falciparum at 8 < t < 16 h. The target of Hit A was searched by immobilising the molecule on a solid support via a linker and performing affinity chromatography on a plasmodial lysate. Several anchoring positions of the linker (6,7 and 3') and PEG-type linkers were assessed, to obtain a linked-hit molecule displaying in vitro antiplasmodial activity similar to that of unmodified Hit A. This allowed us to identify the PfPYK-1 kinase and the PfRab6 GTP-ase as potential targets of Hit A.

Binding studies of promethazine and its metabolites with human serum albumin by high-performance affinity chromatography and molecular docking in the presence of codeine.

"Purple Drank", a soft drink containing promethazine (PMZ) and codeine (COD), has gained global popularity for its hallucinogenic effects. Consuming large amounts of this combination can lead to potentially fatal events. The binding of these drugs to plasma proteins can exacerbate the issue by increasing the risk of drug interactions, side effects, and/or toxicity. Herein, the binding affinity to human serum albumin (HSA) of PMZ and its primary metabolites [N-desmethyl promethazine (DMPMZ) and promethazine sulphoxide (PMZSO)], along with COD, was investigated by high-performance affinity chromatography (HPAC) though zonal approach. PMZ and its metabolites exhibited a notable binding affinity for HSA (%b values higher than 80%), while COD exhibited a %b value of 65%. To discern the specific sites of HSA to which these compounds were bound, displacement experiments were performed using warfarin and (S)-ibuprofen as probes for sites I and II, respectively, which revealed that all analytes were bound to both sites. Molecular docking studies corroborated the experimental results, reinforcing the insights gained from the empirical data. The in silico data also suggested that competition between PMZ and its metabolites with COD can occur in both sites of HSA, but mainly in site II. As the target compounds are chiral, the enantioselectivity for HSA binding was also explored, showing that the binding for these compounds was not enantioselective.

Purification and characterization of thioredoxin reductase enzyme from commercial Spirulina platensis tablets by affinity chromatography and investigation of the effects of some chemicals and drugs on enzyme activity.

Thioredoxin reductase (TrxR, enzyme code [E.C.] 1.6.4.5) is a widely distributed flavoenzyme that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of thioredoxin and many other physiologically important substrates. Spirulina platensis is a blue-green algae that is often used as a dietary supplement. S. platensis is rich in protein, lipid, polysaccharide, pigment, carotenoid, enzyme, vitamins and many other chemicals and exhibits a variety of pharmacological functions. In the present study, a simple and efficient method to purify TrxR from S. platensis tablets is reported. The extractions were carried out using two different methods: heat denaturation and 2',5'-adenosine diphosphate Sepharose 4B affinity chromatography. The enzyme was purified by 415.04-fold over the crude extract, with a 19% yield, and specific activity of 0.7640 U/mg protein. Optimum pH, temperature and ionic strength of the enzyme activity, as well as the Michaelis constant (Km ) and maximum velocity of enzyme (Vmax ) values for NADPH and 5,5'-dithiobis(2-nitrobenzoic acid) were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag+ , Cu2+ , Mg2+ , Ni2+ , Pb2+ , Zn2+ , Al3+ , Cr3+ , Fe3+ , and V4+ ions; vitamin B3 , vitamin B6 , vitamin C, and vitamin U and aciclovir, azithromycin, benzyladenine, ceftriaxone sodium, clarithromycin, diclofenac, gibberellic acid, glurenorm, indole-3-butyric acid, ketorolac, metformin, mupirocin, mupirocin calcium, paracetamol, and tenofovir had inhibitory effects on TrxR. Ag+ exhibited stronger inhibition than 1-chloro-2,4-dinitrobenzene (a positive control).

Development of a new affinity chromatography method for purification of horseradish peroxidase enzyme.

In this study, benzohydroxamic acid molecules were synthesized from methyl 4-amino-2-methoxy, methyl 4-amino-3-nitro, methyl 4-amino-3-methyl, and methyl 4-amino-3-chloro benzoate molecules, and the horseradish peroxidase (HRP) enzyme was purified in one step using the affinity chromatography technique for the first time. The IC50 and Ki values for the 4-amino 3-methyl benzohydroxamic acid molecule were 0.136 and 0.132 ± 0.054 µM, respectively, while the IC50 and Ki values for the 4-amino-3-nitro benzohydroxamic acid molecule were 56.00 and 51.90 ± 9.90 µM, respectively. It was found that the IC50 and Ki values for the 4-amino-3-chloro benzohydroxamic acid molecule were 218.33 and 175.67 ± 43.78 µM, respectively, whereas the IC50 and Ki values for the 4-amino-2-methoxy benzohydroxamic acid molecule were 306.00 and 218.00 ± 68.80 µM, respectively. The HRP enzyme was synthesized from 4-amino-2-methoxy hydroxamic acid column with a 35.97% yield 601.13 times, 4-amino-3-nitro hydroxamic acid column, with a 14.00% yield 404.11 times, 4-amino-3-methyl hydroxamic acid column with an 8.70% yield 394.88 times, and 4-amino-3-chloro hydroxamic acid column with a 4.48% yield 284.85 times. Thus, the HRP enzyme was purified in a single step with hydroxamic acids, and its molecular weight was found to be 44 kDa. The optimum pH was 8.0, the optimum temperature was 15°C, and the optimum ionic strength was 0.4 M for the purified HRP enzyme.

Combination of click chemistry and Schiff base reaction: Post-synthesis of covalent organic frameworks as an immobilized metal ion affinity chromatography platform for efficient capture of global phosphopeptides in serum with chronic obstructive pulmonary disease.

Reasonable design and construction of functionalized materials are of great importance for the enrichment of global phosphopeptides. In this work, Ti4+ functionalized hydrophilic covalent organic frameworks by introducing glutathione (GSH) and 2,3,4-trihydroxy benzaldehyde (THBA) via click chemistry and Schiff base reaction (COF-V@GSH-THBA-Ti4+ ) was constructed and applied for selective enrichment of phosphopeptides in serum. Benefit from the high surface area, excellent hydrophilicity as well as regular mesoporous structure, COF-V@GSH-THBA-Ti4+ displayed high selectivity (molar ratio of 2000:1), low limit of detection (0.5 fmol), high load capacity (100.0 mg/g) and excellent size-exclusion effect (1:10000) for enrichment of phosphopeptides. For actual bio-sample analysis, 15 phosphopeptides assigned to 10 phosphoproteins with 16 phosphorylated sites and 33 phosphopeptides assigned to 25 phosphoproteins with 34 phosphorylated sites were detected from the serum of patients with chronic obstructive pulmonary disease (COPD), and normal controls. Biological processes and molecular functions analysis further disclosed the difference of serums with phosphoproteomics between COPD and normal controls.

Expression, purification, and biological activity evaluation of cathepsin L in mammalian cells.

Cathepsin L (CTSL) could cleave and activate SARS-CoV-2 Spike protein to promote viral entry, making it a hopeful therapeutic target for COVID-19 prevention and treatment. So CTSL inhibitors are considered to be a promising strategy to SARS-CoV-2 infection. CTSL has previously been expressed in inclusion body in Escherichia coli. In order to prepare CTSL with high purity and activity in soluble active form, we transformed HEK-293T cells with a recombinant mammalian expression plasmid. CTSL was purified to a purity about 95%, found to migrate at approximately 43 kDa and exhibited substrate specificity against Z-Phe-Arg-AMC with specific activity of no less than 85 081 U/mg, characteristic of active CTSL. Although eukaryotic purified CTSL is commercially available, our study for the first time reported the details of the expression, purification, and characterization of active, recombinant CTSL in eukaryocyte system, which laid an experimental foundation for the establishment of high-throughput screening model for anti-coronavirus drugs targeting CTSL.

Immobilization of controlled Pu:Am ratio on actinide-specific affinity monolith support developed in capillary and coupled to inductively coupled plasma mass spectrometry.

The objective of this work was to develop an actinide-specific monolithic support in capillary designed to immobilize precise Pu:Am ratios and its coupling to inductively coupled plasma mass spectrometry (ICP-MS) for immobilized metal affinity chromatography applications. This format offers many advantages, such as reducing the sample amount and waste production, which are of prime importance when dealing with highly active radioelements. Four organic phosphorylated-based monoliths were synthesized in situ through UV photo-polymerization in capillary and characterized. The capillary coupling to ICP-MS was set up in conventional laboratory using Th and Sm as chemical analogues of Pu and Am. A dedicated method was developed to quantify online Th and Sm amounts immobilized on the monolithic capillaries, allowing to select the best monolith candidate poly(BMEP-co-EDMA)adp. By precisely adjusting the elemental composition in the loading solutions and applying the developed quantification method, the controlled immobilization of several Th:Sm molar ratios onto the monolith was successful. Finally, the capillary ICP-MS coupling was transposed in a glove box and by applying the strategy developed to design the monolithic support using Th and Sm, the immobilization of a 10.5 ± 0.2 (RSD = 2.3%, n = 3) Pu:Am molar ratio reflecting Pu ageing over 48 years was achieved in a controlled manner on poly(BMEP-co-EDMA)adp. Hence, the new affinity capillary monolithic support was validated, with only hundred nanograms or less of engaged radioelements and can be further exploited to precisely determine differential interactions of Pu and Am with targeted biomolecules in order to better anticipate the effect of Am on Pu biodistribution.

Molecular Targets of the 5-Amido-Carboxamide Bumped Kinase Inhibitor BKI-1748 in Cryptosporidium parvum and HCT-8 Host Cells.

Cryptosporidium parvum is an apicomplexan parasite causing persistent diarrhea in humans and animals. Issuing from target-based drug development, calcium-dependent protein kinase 1 inhibitors, collectively named bumped kinase inhibitors (BKIs), with excellent efficacies in vitro and in vivo have been generated. Some BKIs including BKI-1748 share a core structure with similarities to the first-generation antiprotozoal drug quinine, which is known to exert notorious side effects. Unlike quinine, BKI-1748 rapidly interfered with C. parvum proliferation in the human colon tumor (HCT) cell line HCT-8 cells and caused dramatic effects on the parasite ultrastructure. To identify putative BKI targets in C. parvum and in host cells, we performed differential affinity chromatography with cell-free extracts from non-infected and infected HCT-8 cells using BKI-1748 and quinine epoxy-activated sepharose columns followed by mass spectrometry. C. parvum proteins of interest were identified in eluates from columns coupled to BKI-1748, or in eluates from both BKI-1748 and quinine columns. However, no C. parvum proteins could be identified binding exclusively to BKI-1748. In contrast, 25 BKI-1748-specific binding proteins originating from HCT-8 cells were detected. Moreover, 29 C. parvum and 224 host cell proteins were identified in both BKI-1748 as well as in quinine eluates. In both C. parvum and host cells, the largest subset of binding proteins was involved in RNA binding and modification, with a focus on ribosomal proteins and proteins involved in RNA splicing. These findings extend previous results, showing that BKI-1748 interacts with putative targets involved in common, essential pathways such as translation and RNA processing.