Establishment and application of a screening method for α-glucosidase inhibitors based on dual sensing and affinity chromatography.

α-Glucosidase plays a direct role in the metabolic pathways of starch and glycogen, any dysfunction in its activity could result in metabolic disease. Concurrently, this enzyme serves as a target for diverse drugs and inhibitors, contributing to the regulation of glucose metabolism in the human body. Here, an integrated analytical method was established to screen inhibitors of α-glucosidase. This step-by-step screening model was accomplished through the biosensing and affinity chromatography techniques. The newly proposed sensing program had a good linear relationship within the enzyme activity range of 0.25 U mL-1 to 1.25 U mL-1, which can quickly identify active ingredients in complex samples. Then the potential active ingredients can be captured, separated, and identified by an affinity chromatography model. The combination of the two parts was achieved by an immobilized enzyme technology and a microdevice for reaction, and the combination not only ensured efficiency and accuracy for inhibitor screening but also eliminated the occurrence of false positive results in the past. The emodin, with a notable inhibitory effect on α-glucosidase, was successfully screened from five traditional Chinese medicines using this method. The molecular docking results also demonstrated that emodin was well embedded into the active pocket of α-glucosidase. In summary, the strategy provided an efficient method for developing new enzyme inhibitors from natural products.

Purification of pineapple bromelain by IMAC chromatography using chlorophyll-activated macroporous matrices.

This study investigated the purification of bromelain obtained from pineapple fruit using a new adsorbent for immobilized metal ion affinity chromatography (IMAC), with chlorophyll obtained from plant leaves as a chelating agent. The purification of bromelain was evaluated in batches from the crude extract of pineapple pulp (EXT), and the extract precipitated with 50 % ammonium sulfate (EXT.PR), the imidazole buffer (200 mM, pH 7.2) being analyzed and sodium acetate buffer, pH 5.0 + 1.0 NaCl as elution solutions. All methods tested could separate forms of bromelain with molecular weights between ±21 to 25 kDa. Although the technique using EXT.PR stood out in terms of purity, presenting a purification factor of around 3.09 ± 0.31 for elution with imidazole and 4.23 ± 0.12 for acetate buffer solution. In contrast, the EXT methods obtained values between 2.44 ± 0.23 and 3.21 ± 0.74 for elution with imidazole and acetate buffer, respectively, for purification from EXT.PR has lower yield values (around 5 %) than EXT (around 15 %). The number of steps tends to reduce yield and increase process costs, so the purification process in a monolithic bed coupled to the chromatographic system using the crude extract was evaluated. The final product obtained had a purification factor of 6, with a specific enzymatic activity of 59.61 ± 0.00 U·mg-1 and a yield of around 39 %, with only one band observed in the SDS-PAGE electrophoresis analysis, indicating that the matrix produced can separate specific proteins from the total fraction in the raw material. The IMAC matrix immobilized with chlorophyll proved promising and viable for application in protease purification processes.

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).

Magnetic Nanoparticles for Protein Separation and Purification.

Proteins are essential for various functions such as brain activity and muscle contraction in humans. Even though food is a source of proteins, the bioavailability of proteins in most foods is usually limited due to matrix interaction with other biomolecules. Thus, it is essential to extract these proteins and provide them as a nutraceutical supplement to maintain protein levels and avoid protein deficiency. Hence, protein purification and extraction from natural sources are highly significant in biomedical applications. Chromatography, crude mechanical disruption, use of extractive chemicals, and electrophoresis are some of the methods applied to isolate specific proteins. Even though these methods possess several advantages, they are unable to extract specific proteins with high purity. A suitable alternative is the use of nanoparticles, which can be beneficial in protein purification and extraction. Notably, magnetic iron and iron-based nanoparticles have been employed in protein extraction processes and can be reused via demagnetization due to their magnetic property, smaller size, morphology, high surface-to-volume ratio, and surface charge-mediated property. This chapter is a summary of various magnetic nanoparticles (MNPs) that can be used for the biomolecular separation of proteins.

Multi-target bioactive compound screening from the infructescence of Platycarya strobilacea Sieb. et Zucc. by affinity chromatography using immobilized ß2 -adrenoceptor and muscarinic-3 acetylcholine receptor as the stationary phase.

As a main source for the recognition and identification of lead compounds, traditional Chinese medicine plays a pivotal role in preventing diseases for years. However, screening bioactive compounds from traditional Chinese medicine remains challenging because of the complexity of the systems and the occurrence of the synergic effect of the compounds. The infructescence of Platycarya strobilacea Sieb. et Zucc is prescribed for allergic rhinitis treatment with unknown bioactive compounds and unclear mechanisms. Herein, we immobilized the ß2 -adrenoceptor and muscarine-3 acetylcholine receptor onto the silica gel surface to prepare the stationary phase in a covalent bond through one step. The feasibility of the columns was investigated by the chromatographic method. Ellagic acid and catechin were identified as the bioactive compounds targeting the receptors. The binding constants of ellagic acid were calculated to be (1.56 ± 0.23)×107  M-1 for muscarine-3 acetylcholine receptor and (2.93 ± 0.15)×107  M-1 for ß2 -adrenoceptor by frontal analysis. While catechin can bind with muscarine-3 acetylcholine receptor with an affinity of (3.21 ± 0.05)×105  M-1 . Hydrogen bonds and van der Waals' force were the main driving forces for the two compounds with the receptors. The established method provides an alternative for multi-target bioactive compound screening in complex matrices.

Development of two immunochromatographic test strips based on signal amplification and selenium nanoparticles for the rapid detection of T-2 mycotoxin.

Conventional immunochromatographic test strips (ICSs) based on gold nanoparticle (AuNP) probes offer limited sensitivity. Here, AuNPs were separately labeled with monoclonal or secondary antibodies (MAb or SAb). In addition, spherical, homogeneously dispersed, and stable selenium nanoparticles (SeNPs) were also synthesized. By optimizing the preparation parameters, two ICSs based on the dual AuNP signal amplification (Duo-ICS) or SeNPs (Se-ICS) were developed for the rapid detection of T-2 mycotoxin. The detection sensitivities of the Duo-ICS and Se-ICS assays for T-2 were 1 ng/mL and 0.25 ng/mL, respectively, which were 3-fold and 15-fold more sensitive, respectively, than a conventional ICS. Furthermore, the ICSs were applied in the detection of T-2 in cereals, which requires higher sensitivity. Our findings indicate that both ICS systems can be used for rapid, sensitive, and specific detection of T-2 toxin in cereals and potentially other sample types.

Magnetic Ti3C2 MXene Nanosheets Prepared for Enrichment of Phosphopeptides.

MXenes have received lots of attention since discovered and have been applied in various fields. In this work, Ti3C2-Fe3O4 composites with exposed non-modified Ti3C2 MXene nanosheets were designed and prepared by an in situ growth strategy and then applied in the enrichment of phosphopeptides. The two-dimensional composites could interact with the phosphopeptides through a metal oxide affinity chromatography mechanism provided by Ti-O and Fe-O bonds and a hydrophilic interaction chromatography mechanism by surface hydroxyl groups. This magnetic nanomaterial with a specific surface area of 66.1 m2·g-1 had high sensitivity to phosphopeptides (0.5 nmol·L-1) and high selectivity (1:1000 of the molar ratio of ß-casein to bovine serum albumin). Non-fat milk was adopted as a real sample to preliminarily examine the applicability of the Ti3C2-Fe3O4-based protocol. Subsequently, Qingkailing injection, a kind of traditional Chinese medicine injection, was introduced to further explore the suitability of the nanocomposites for phosphopeptide enrichment from more complex matrices and satisfactory results were obtained.

Rapid separation and binding configuration prediction of the components in Danshen decoction to endothelin A receptor using affinity chromatography and molecular dynamics simulation.

As a famous traditional Chinese formula, Danshen Decoction has the potential to relieve the pain of pulmonary arterial hypertension patients, however, the functional components remain unknown. Herein, we reported a method to screen the functional components in Danshen Decoction targeting endothelin receptor A, an accepted target for the treatment of the disease. The receptor was functionalized on the macroporous silica gel through an epidermal growth factor receptor fusion tag and its covalent inhibitor. Using the affinity gel as the stationary phase, the bioactive compound was identified as salvianolic acid B by mass spectrometry. The binding kinetic parameter (dissociation rate constants kd ) of salvianolic acid B with the receptor was determined via peak profiling. Using the specific ligands of the receptor as probes, the binding configuration prediction of salvianolic acid B with the receptor was performed by molecular dynamics simulation. Our results indicated that salvianolic acid B is a potential bioactive compound in Danshen Decoction targeting the receptor. This work showed that receptor chromatography in combination with molecular dynamics simulation is applicable to predicting the binding kinetics and configuration of a ligand to a receptor, providing crucial insight for the rational design of drugs that recognize functional proteins.

Towards the in vivo identification of protein-protein interactions.

Protein-protein interactions (PPIs) play crucial roles in biological processes. The conventional methods based on affinity purification of a protein of interest (POI) have been widely used to identify unknown PPIs. Recently, proximity-dependent biotin identification (BioID) has been used increasingly to investigate PPIs. BioID utilizes the proximity-dependent biotinylation, in the presence of biotin, of endogenous proteins that are located within a certain distance of POI-fused biotin ligase, which enables us to reveal the more physiologically relevant PPIs in vivo compared to the conventional methods. However, there is little information on an appropriate way to administer biotin in vivo. Recent studies reported some biotin supplementations for in vivo BioID. In this commentary, we review the BioID technique as a tool to examine PPIs, and we introduce a potential method to achieve efficient proximity labelling for in vivo BioID.

Stepwise Frontal Analysis Coupled with Affinity Chromatography: A Fast and Reliable Method for Potential Ligand Isolation and Evaluation from Mahuang-Fuzi-Xixin Decoction.

Mahuang-Fuzi-Xixin Decoction (MFXD) is widely used in the treatment of asthma, however, the functional components in the decoction targeting beta2-adrenoceptor (ß2 -AR) remain unclear. Herein, we immobilized the haloalkane dehalogenase (Halo)-tagged ß2 -AR on the 6-chlorocaproic acid-modified microspheres. Using the affinity stationary phase, the interactions of four ligands with the receptor were analyzed by stepwise frontal analysis. The association constants were (4.75±0.28)×104  M-1 for salbutamol, (2.93±0.15)×104  M-1 for terbutaline, (1.23±0.03)×104  M-1 for methoxyphenamine, (5.67±0.38)×104  M-1 for clorprenaline at high-affinity binding site, and (2.73±0.05)×103  M-1 at low-affinity binding site. These association constants showed the same rank order as the radioligand binding assay, demonstrating that immobilized ß2 -AR had capacity to screen bioactive compounds binding to the receptor while stepwise frontal analysis could predict their binding affinities. Application of the immobilized receptor in analysis of MFXD by chromatographic method revealed that ephedrine, aconifine, karakoline, and chasmanine were the bioactive compounds targeting ß2 -AR. Among them, ephedrine and chasmanine exhibited association constants of (2.94±0.02)×104 M-1 and (4.60±0.15)×104  M-1 to the receptor by stepwise frontal analysis. Molecular docking analysis demonstrated that ephedrine, chasmanine, and the other two compounds interact with ß2 -AR through the same pocket involving the key amino acids such as Asn312, Asp113, Phe289, Trp286, Tyr316, and Val114. As such, we reasoned that the four compounds dominate the therapeutic effect of MFXD against asthma through ß2 -AR mediating pathway. This work shed light on the potential of immobilized ß2 -AR for drug discovery and provided a valuable methodology for rapid screening.

Developing an ultrasensitive immunochromatographic assay for authentication of an emergent fraud aminopyrine in herbal tea.

Aminopyrine is a nonsteroidal anti-inflammatory drug only for medical purposes, however, it has been illegally added in traditional Chinese herbal teas for fraud activity recently. In this study, a specific antibody against aminopyrine with IC50 of 3.00 ng/mL was obtained for the first time by a rational hapten design. Furthermore, an ultrasensitive gold nanoparticles immunochromatographic assay (AuNPs-ICA) for determination of aminopyrine based on a portable reader was firstly developed, with cut-off value of 100.00 ng/mL, limit of detection (LOD) of 4.80 ng/mL and limit of quantification (LOQ) of 5.71 ng/mL for herbal tea, respectively. The recovery rates ranged from 93.21 % to 105.61 %, with inter-assay coefficient of variation (CV) from 1.08 % to 3.82 %. Additionally, 24 blind samples were examined simultaneously by AuNPs-ICA and LC-MS/MS, demonstrating a good consistency for each other. The proposed AuNPs-ICA is an ultrasensitive and reliable tool for on-site surveillance screening of fraud additives in herbal tea.

Rapid and sensitive quantification of capsaicinoids for edible oil adulteration by immunomagnetic solid-phase extraction coupled with time-resolved fluorescent immunochromatographic assay.

Immunochromatographic methods are acknowledged analytic assay to analyze capsaicinoids. Immunomagnetic solid-phase extraction (IMSPE) coupled with time-resolved fluorescence immunochromatographic assay (TRFICA) was proposed to quantify capsaicinoids in oil samples. Monoclonal antibodies (mAb) were synthesized with CNBr-Magnetic Crystarose 4B particles (CNBr-MCPs) under mild condition. The resultant CNBr-MCPs@mAb were conjugated high affinity mAbs on its surface, which was utilized to extract capsaicinoids from lipid matrices via antibodies-antigens capture. Under the optimized conditions, the whole IMSPE procedure was achieved within 15 min, and quantified by TRFICA strips. The results showed coefficients up to 0.9975 and the visual detection limit as low as 0.6 µg kg-1. The recoveries were ranging from 88.3 % to 112.4 % with the intra-day and inter-day precision lower than 11.6 %. Finally, the proposed IMSPE-TRFICA method was successfully used to detect capsaicinoids in lipid matrices, which has great utility to quantify capsaicinoids and adulteration detect vegetable oils.

Rapid screening of α-glucosidase inhibitors in Hypericum perforatum L. using bio-affinity chromatography coupled with UPLC/MS.

α-glucosidase inhibitors (AGIs) are widely used for the treatment of type 2 diabetes, but their side effects have made it to develop novel and alternative AGIs immediately. In this study, the extract of Hypericum perforatum L. (HPE) has been confirmed to have α-glucosidase inhibitory activity in vitro and in vivo. Seven active compounds, rutin, hyperoside, isoquercitrin, avicularin, quercitrin, quercetin, and biapigenin, were screened based on a bio-affinity chromatography column with α-glucosidase enzyme-conjugated solid phase and UPLC/MS, which exhibited excellent α-glycosidase inhibitory effects by the determined IC50 values. The mechanism of α-glycosidase inhibitory activity of biapigenin was studied for the first time. The results showed that biapigenin was a high-potential, reversible, and mixed enzyme inhibitor. Analysis by molecular docking further revealed that hydrophobic interactions were generated by interactions between biapigenin and amino acid residues LYS156, PHE303, PHE314, and LEU313. In addition, hydrogen bonding occurred between biapigenin and α-glucosidase amino acid residues ASP307, SER241, and LYS156. This research identified that biapigenin could be a novel AGI and further applied to the development of potential anti-diabetic drugs. Furthermore, our studies established a rapid in vitro screening method for AGIs from plants.

Tandem immuno-purification of affinity-tagged proteins from mouse testis extracts for MS analysis.

Here, we describe a protocol for extraction and tandem immunoprecipitation of the cytoplasmic and nuclear proteins from mouse testis for mass spectrometry. This protocol has been applied to knockin mice that express a meiotic protein of interest tagged with 3xFLAG-HA in the testis. The protocol is optimized for salt extraction of cytoplasmic and nuclear proteins from mouse frozen testes and thus can be used for a variety of proteins. For complete details on the use and execution of this protocol, please refer to Ishiguro et al. (2020) and Tanno et al. (2022).

Activated Carbon-Based Immunochromatographic Strip Test for the Rapid Qualitative Analysis of Swertiamarin and Sweroside.

BACKGROUND: Swertia japonica (S. japonica) is a medicinal plant that belongs to the Gentianaceae family. Several reports confirm the biological effects of the S. japonica extract. This plant is used mainly as a digestive stimulant, appetite stimulant, and gastrointestinal disease remedy in Japan. Secoiridoid glycosides are a group of compounds related to the beneficial effects of this plant. OBJECTIVE: We developed an immunochromatographic strip test for major secoiridoid glycosides, such as swertiamarin (SM) and sweroside (SS) detection. METHODS: We fabricated an immunoprobe using activated carbon as a reporter molecule and a monoclonal antibody against SM and SS (MAb D2) as a detection molecule. The test and control zones of the strip test contained SM-cBSA and Goat pAb anti-mouse IgM HRP conjugate, respectively. The immunoprobe reacted competitively with free SM and/or SS and immobilized SM-cBSA. The results were read and interpreted by the black spot intensity in the test zone. RESULTS: We succeeded in developing a strip test system with a detection limit (LOD) of 12.5 µg/mL. The selectivity and reliability evaluation revealed that the strip test is suitable for detecting SM and SS in S. japonica. The result was ready to be read in 30 min. CONCLUSIONS: This method can be a useful tool for the screening of biologically active S. japonica samples for further preparation of traditional medicine. HIGHLIGHTS: To the best of our knowledge, this is the first immunochromatographic strip test developed for the detection of SM and SS in S. japonica samples.

Rapid and sensitive detection of tert-butylhydroquinone in soybean oil using a gold-based paper sensor.

tert-Butylhydroquinone (TBHQ) residues in foods pose a threat to human health. Therefore, it is necessary to develop a rapid method for TBHQ detection. In this study, a sensitive monoclonal antibody 5C3 (IgG2a subclass) against TBHQ was produced. It possessed a half maximal inhibitory concentration of 7.43 ng mL-1. A gold nanoparticle-based immunochromatographic assay (ICA) was established for the rapid and sensitive screening of TBHQ in soybean oil. Qualitative analysis results were obtained within 10 min and observed with the naked eye. The visual limit of detection (LOD) was 50 ng g-1 and the cut-off value was 1000 ng g-1. A hand-held strip reader was used for quantitative analysis, in which the calculated LOD was defined as 18.68 ng g-1. The average recoveries of TBHQ ranged from 89.55% ± 2.70% to 100.66% ± 3.02% for soybean oil, with a coefficient of variation of 2.89%-7.05%. Therefore, our developed ICA is a useful tool for the rapid and on-site detection of TBHQ in real food samples.

Purification of damson plum polyphenol oxidase by affinity chromatography and investigation of metal effects on enzyme activity.

Polyphenol oxidase (PPO) was firstly purified from damson plum as a high antioxidant source. PPO was treated by 0-80% ammonium sulfate precipitation and dialysis. Characterization results were determined for catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05 M/pH: 7.2/25 °C; 0.2 M/pH: 4.5/10 °C; 0.01 M/pH: 6.8/5 °C, and 0.2 M/pH: 8.5/10 °C, respectively. Vmax and KM values were calculated for same substrates as 17,219.97 U/(mL*min) and 11.67 mM; 7309.72 U/(mL*min) and 5 mM; 12,580.12 U/(mL*min) and 3.74 mM; 12,100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest Vmax value among substrates. Affinity purification was performed by using Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose 6B-L-Tyrosine-p-aminobenzoic acid. Single bands were approximately observed at 50 kDa for each affinity sample in SDS-PAGE and Native-PAGE. 93.88 and 10.46 purification-folds were obtained for PPO by reference Sepharose-4B and original Sepharose-6B gels. Metal effects upon PPO activity were also investigated due to the importance of enzymatic browning in foods. Cu+2 activation and Fe+2 inhibition were observed with a final metal concentration of 1 mM at 219.66 and 43.18%, respectively. PPO purification from damson plum by affinity chromatography, its characterization, stability evaluation by statistically, and effects of metal ions on damson plum PPO have not been investigated in the literature.

Broad-specific immunochromatography for simultaneous detection of various sulfonylureas in adulterated multi-herbal tea.

Sulfonylureas (SUs) are a series of anti-diabetic drugs widely used for type 2 diabetes mellitus for clinic treat. However, it is often illegally adulterated in multi-herbal tea to improve the claimed anti-diabetic activity in recent years. In this study, a novel hapten was rationally designed, and a broad-specific monoclonal antibody (anti-SUs mAb) recognizing nine SUs was developed. This mAb was used to develop a colloidal gold lateral flow immunochromatographic assay (CG-LFIA). The anti-SUs mAb demonstrated half inhibitory concentration (IC50) ranged from 0.15 ng/mL to 3.25 ng/ mL for nine SUs by ELISA. The cut-off value of developed CG-LFIA for nine SUs was from 3 to 100 ng/ mL for the spiked samples. LC-MS/MS confirmed the reliability of the new CG-LFIA. The results indicated that the proposed CG-LFIA could be an ideal method in on-site screening surveillance assay for SUs illegally adulterated in multi-herbal tea products.

Targeted Separation of COX-2 Inhibitor from Pterocephalus hookeri Using Preparative High-Performance Liquid Chromatography Directed by the Affinity Solid-Phase Extraction HPLC System.

Pterocephalus hookeri, as a kind of popular traditional Tibetan medicine, is reputed to treat inflammatory related diseases. In the present work, a cyclooxygenase-2 functionalized affinity solid-phase extraction HPLC system was developed and combined with preparative-HPLC for rapidly screening and separating cyclooxygenase-2 ligand from P. hookeri extracts. Firstly, ligands of cyclooxygenase-2 were screened from extracts by affinity solid-phase extraction HPLC system. Then directed by the screening results, the recognized potential active compounds were targeted separated. As a result, the major cyclooxygenase-2 inhibitor of P. hookeri was obtained with a purity of >95%, which was identified as sylvestroside I. To test the accuracy of this method, the anti-inflammatory activity of sylvestroside I was inspected in lipopolysaccharide-induced RAW 264.7 cells. The results show that sylvestroside I significantly suppressed the release of prostaglandin E2 with dose-dependent, which was in good agreement with the screening result of the affinity solid-phase method. This method of integration of screening and targeted separation proved to be very efficient for the recognition and isolation of cyclooxygenase-2 inhibitors from natural products.

Characterization of Cytotoxic Lactose Binding Lectin from Sulphur Polypore, Laetiporus sulphureus (Agaricomycetes), from Algeria.

Mushroom lectins have important biological and biomedical applications. Most lectins purified from these organisms exhibit high toxicity in animal cells and toward microbial agents. They are able to induce cell growth inhibition and metabolism by their ability to interact with glyconjugate components (glycoproteins receptors, glycolipids) present in their membrane. After lectins bind to these membrane receptors, they induce cellular signalization chains in which gene expression is regulated and cell death programming (apoptosis) is activated. In this work, a new multimeric lectin was characterized from the rare saprobic edible mushroom, Laetiporus sulphureus strain TMES43, grown in the Algerian forest. Lectin was isolated with ammonium sulfate precipitation followed by affinity chromatography on a Sepharose 4B column, with specific activity of 1204.7 units of hemagglutination activity/mg and 35.55% yield. The protein has a tetrameric structure with a molecular weight of 36 kDa for each subunit, with a total molecular weight of approximately 140 kDa. In addition, a Mascot peptide fingerprint study on a matrix-assisted laser desorption ionization-time of flight tandem fragment showed identity with autophagy-related protein 16 from Meyerozyma guilliermondii (strain ATCC 6260/CBS 566/DSM 6381/JCM 1539/NBRC 10279/NRRL Y-324; Expasy ID: ATG16_PICGU) and no sequence similarity to known mushroom lectins. L. sulphureus hemagglutination activity was reduced by 5 mM of lactose and 10 mM of EDTA incubation and was recovered by metallic cations such as CaCl2, MgCl2, and ZnCl2. L. sulphureus purified lectin had no human ABO group specificity and showed low temperature and alkaline pH stabilities. The MTT preliminary assay showed that L. sulphureus purified lectin induced high cytotoxicity for tumor cells and normal cells.