Displaying publications 1 - 20 of 56 in total

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  1. A simple and high efficiency purification of His-tagged turnip yellow mosaic virus-like particle (TYMV-VLP) by nickel ion affinity precipitation
    Tan FH, Ng JF, Mohamed Alitheen NB, Muhamad A, Yong CY, Lee KW
    J Virol Methods, 2023 Sep;319:114771.
    PMID: 37437780 DOI: 10.1016/j.jviromet.2023.114771
    Virus-like particles (VLPs) is one of the most favourable subjects of study, especially in the field of nanobiotechnology and vaccine development because they possess good immunogenicity and self-adjuvant properties. Conventionally, VLPs can be tagged and purified using affinity chromatography or density gradient ultracentrifugation which is costly and time-consuming. Turnip yellow mosaic virus (TYMV) is a plant virus, where expression of the viral coat protein (TYMVc) in Escherichia coli (E. coli) has been shown to form VLP. In this study, we report a non-chromatographic method for VLP purification using C-terminally His-tagged TYMVc (TYMVcHis6) as a protein model. Firstly, the TYMVcHis6 was cloned and expressed in E. coli. Upon clarification of cell lysate, nickel (II) chloride [NiCl2; 15 µM or equivalent to 0.0000194% (w/v)] was added to precipitate TYMVcHis6. Following centrifugation, the pellet was resuspended in buffer containing 1 mM EDTA to chelate Ni2+, which is then removed via dialysis. A total of 50% of TYMVcHis6 was successfully recovered with purity above 0.90. Later, the purified TYMVcHis6 was analysed with sucrose density ultracentrifugation, dynamic light scattering (DLS), and transmission electron microscopy (TEM) to confirm VLP formation, which is comparable to TYMVcHis6 purified using the standard immobilized metal affinity chromatography (IMAC) column. As the current method omitted the need for IMAC column and beads while significantly reducing the time needed for column washing, nickel affinity precipitation represents a novel method for the purification of VLPs displaying poly-histidine tags (His-tags).
    Matched MeSH terms: Chromatography, Affinity/methods
  2. Identification and Quantification of Affinity-Purified Proteins with MaxQuant, Followed by the Discrimination of Nonspecific Interactions with the CRAPome Interface
    Lee PY, Low TY
    Methods Mol Biol, 2023;2690:299-310.
    PMID: 37450156 DOI: 10.1007/978-1-0716-3327-4_25
    Affinity purification coupled to mass spectrometry (AP-MS) is a powerful method to analyze protein-protein interactions (PPIs). The AP-MS approach provides an unbiased analysis of the entire protein complex and is useful to identify indirect interactors. However, reliable protein identification from the complex AP-MS experiments requires appropriate control of false identifications and rigorous statistical analysis. Another challenge that can arise from AP-MS analysis is to distinguish bona fide interacting proteins from the non-specifically bound endogenous proteins or the "background contaminants" that co-purified by the bait experiments. In this chapter, we will first describe the protocol for performing in-solution trypsinization for the samples from the AP experiment followed by LC-MS/MS analysis. We will then detail the MaxQuant workflow for protein identification and quantification for the PPI data derived from the AP-MS experiment. Finally, we describe the CRAPome interface to process the data by filtering against contaminant lists, score the interactions and visualize the protein interaction networks.
    Matched MeSH terms: Chromatography, Affinity/methods
  3. Tandem Affinity Purification (TAP) of Interacting Prey Proteins with FLAG- and HA-Tagged Bait Proteins
    Low TY, Lee PY
    Methods Mol Biol, 2023;2690:69-80.
    PMID: 37450137 DOI: 10.1007/978-1-0716-3327-4_6
    Proteins often interact with each other to form complexes and play functional roles in almost all cellular processes. The study of protein-protein interactions is therefore critical to understand protein function and biological pathways. Affinity Purification coupled with Mass Spectrometry (AP-MS) is an invaluable technique for identifying the interaction partners in protein complexes. In this approach, the protein of interest is fused to an affinity tag, followed by the expression and purification of the fusion protein. The affinity-purified sample is then analyzed by mass spectrometry to identify the interaction partners of the bait proteins. In this chapter, we detail the protocol for tandem affinity purification (TAP) based on the use of the FLAG (a fusion tag with peptide sequence DYKDDDDK) and hemagglutinin (HA) peptide epitopes. The immunoprecipitation using dual-affinity tags offers the advantage of increasing the specificity of the purification with lower nonspecific-background interactions.
    Matched MeSH terms: Chromatography, Affinity/methods
  4. Fulltext Recent progress in mass spectrometry-based strategies for elucidating protein-protein interactions
    Low TY, Syafruddin SE, Mohtar MA, Vellaichamy A, A Rahman NS, Pung YF, et al.
    Cell Mol Life Sci, 2021 Jul;78(13):5325-5339.
    PMID: 34046695 DOI: 10.1007/s00018-021-03856-0
    Protein-protein interactions are fundamental to various aspects of cell biology with many protein complexes participating in numerous fundamental biological processes such as transcription, translation and cell cycle. MS-based proteomics techniques are routinely applied for characterising the interactome, such as affinity purification coupled to mass spectrometry that has been used to selectively enrich and identify interacting partners of a bait protein. In recent years, many orthogonal MS-based techniques and approaches have surfaced including proximity-dependent labelling of neighbouring proteins, chemical cross-linking of two interacting proteins, as well as inferring PPIs from the co-behaviour of proteins such as the co-fractionating profiles and the thermal solubility profiles of proteins. This review discusses the underlying principles, advantages, limitations and experimental considerations of these emerging techniques. In addition, a brief account on how MS-based techniques are used to investigate the structural and functional properties of protein complexes, including their topology, stoichiometry, copy number and dynamics, are discussed.
    Matched MeSH terms: Chromatography, Affinity/methods*
  5. Membrane-bound Δ12 fatty acid desaturase (FAD12); From Brassica napus to E. coli expression system
    Halim NFAA, Ali MSM, Leow ATC, Rahman RNZRA
    Int J Biol Macromol, 2021 Jun 01;180:242-251.
    PMID: 33737181 DOI: 10.1016/j.ijbiomac.2021.03.072
    Fatty acid desaturase catalyzes the desaturation reactions by insertion of double bonds into the fatty acyl chain, producing unsaturated fatty acids. Though soluble fatty acid desaturases have been studied widely in advanced organisms, there are very limited studies of membrane fatty acid desaturases due to the difficulty of generating recombinant desaturase. Brassica napus is a rapeseed, which possesses a range of different membrane-bound desaturases capable of producing fatty acids including Δ3, Δ4, Δ8, Δ9, Δ12, and Δ15 fatty acids. The 1155 bp open reading frame of Δ12 fatty acid desaturase (FAD12) from Brassica napus codes for 383 amino acid residues with a molecular weight of 44 kDa. It was expressed in Escherichia coli at 37 °C in soluble and insoluble forms when induced with 0.5 mM IPTG. Soluble FAD12 has been purified using Ni2+-Sepharose affinity chromatography with a total protein yield of 0.728 mg/mL. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that desaturase activity of FAD12 could produce linoleic acid from oleic acid at a retention time of 17.6 with a conversion rate of 47%. Characterization of purified FAD12 revealed the optimal temperature of FAD12 was 50 °C with 2 mM preferred substrate concentration of oleic acid. Analysis of circular dichroism (CD) showed FAD12 was made up of 47.3% and 0.9% of alpha-helix and β-sheet secondary structures. The predicted Tm value was 50.2 °C.
    Matched MeSH terms: Chromatography, Affinity
  6. Identification of potential glycoprotein biomarkers in oral squamous cell carcinoma using sweet strategies
    Wong YL, Anand R, Yuen KM, Mustafa WMW, Abraham MT, Tay KK, et al.
    Glycoconj J, 2021 02;38(1):1-11.
    PMID: 33547992 DOI: 10.1007/s10719-021-09973-z
    The prevalence of oral squamous cell carcinoma (OSCC) is high in South and Southeast Asia regions. Most OSCC patients are detected at advanced stages low 5-year survival rates. Aberrant expression of glycosylated proteins was found to be associated with malignant transformation and cancer progression. Hence, identification of cancer-associated glycoproteins could be used as potential biomarkers that are beneficial for diagnosis or clinical management of patients. This study aims to identify the differentially expressed glycoproteins using lectin-based glycoproteomics approaches. Serum samples of 40 patients with OSCC, 10 patients with oral potentially malignant disorder (OPMD), and 10 healthy individuals as control group were subjected to two-dimensional gel electrophoresis (2-DE) coupled with lectin Concanavalin A and Jacalin that specifically bind to N- and O-glycosylated proteins, respectively. Five differentially expressed N- and O-glycoproteins with various potential glycosylation sites were identified, namely N-glycosylated α1-antitrypsin (AAT), α2-HS-glycoprotein (AHSG), apolipoprotein A-I (APOA1), and haptoglobin (HP); as well as O-glycosylated AHSG and clusterin (CLU). Among them, AAT and APOA1 were further validated using enzyme-linked immunosorbent assay (ELISA) (n = 120). It was found that AAT and APOA1 are significantly upregulated in OSCC and these glycoproteins are independent risk factors of OSCC. The clinical utility of AAT and APOA1 as potential biomarkers of OSCC is needed for further evaluation.
    Matched MeSH terms: Chromatography, Affinity/methods
  7. Fulltext Selection and characterisation of Affimers specific for CEA recognition
    Shamsuddin SH, Jayne DG, Tomlinson DC, McPherson MJ, Millner PA
    Sci Rep, 2021 01 12;11(1):744.
    PMID: 33436840 DOI: 10.1038/s41598-020-80354-6
    Carcinoembryonic antigen (CEA) is the only blood based protein biomarker at present, used for preoperative screening of advanced colorectal cancer (CRC) patients to determine the appropriate curative treatments and post-surveillance screening for tumour recurrence. Current diagnostics for CRC detection have several limitations and development of a highly sensitive, specific and rapid diagnostic device is required. The majority of such devices developed to date are antibody-based and suffer from shortcomings including multimeric binding, cost and difficulties in mass production. To circumvent antibody-derived limitations, the present study focused on the development of Affimer proteins as a novel alternative binding reagent for CEA detection. Here, we describe the selection, from a phage display library, of Affimers specific to CEA protein. Characterization of three anti-CEA Affimers reveal that these bind specifically and selectively to protein epitopes of CEA from cell culture lysate and on fixed cells. Kinetic binding analysis by SPR show that the Affimers bind to CEA with high affinity and within the nM range. Therefore, they have substantial potential for used as novel affinity reagents in diagnostic imaging, targeted CRC therapy, affinity purification and biosensor applications.
    Matched MeSH terms: Chromatography, Affinity/methods*
  8. Batch and dynamic adsorption of lysozyme from chicken egg white on dye-affinity nanofiber membranes modified by ethylene diamine and chitosan
    Show PL, Ooi CW, Lee XJ, Yang CL, Liu BL, Chang YK
    Int J Biol Macromol, 2020 Nov 01;162:1711-1724.
    PMID: 32805284 DOI: 10.1016/j.ijbiomac.2020.08.065
    Adsorption of lysozyme on the dye-affinity nanofiber membranes was investigated in batch and dynamic modes. The membrane matrix was made of electrospun polyacrylonitrile nanofibers that were grafted with ethylene diamine (EDA) and/or chitosan (CS) for the coupling of Reactive Blue 49 dye. The physicochemical properties of these dye-immobilized nanofiber membranes (P-EDA-Dye and P-CS-Dye) were characterized microscopically, spectroscopically and thermogravimetrically. The capacities of lysozyme adsorption by the dye-affinity nanofiber membranes were evaluated under various conditions, namely pH, dye immobilized density, and loading flow rate. The adsorption of lysozyme to the dye-affinity nanofiber membranes was well fitted by Langmuir isotherm and pseudo-second kinetic models. P-CS-Dye nanofiber membrane had a better performance in the dynamic adsorption of lysozyme from complex chicken egg white solution. It was observed that after five cycles of adsorption-desorption, the dye-affinity nanofiber membrane did not show a significant loss in its capacity for lysozyme adsorption. The robustness as well as high dynamic adsorption capability of P-CS-Dye nanofiber membrane are promising for the efficient recovery of lysozyme from complex feedstock via nanofiber membrane chromatography.
    Matched MeSH terms: Chromatography, Affinity*
  9. Effective purification of lysozyme from chicken egg white by tris(hydroxymethyl)aminomethane affinity nanofiber membrane
    Liu BL, Ooi CW, Ng IS, Show PL, Lin KJ, Chang YK
    Food Chem, 2020 Oct 15;327:127038.
    PMID: 32447136 DOI: 10.1016/j.foodchem.2020.127038
    Polyacrylonitrile nanofiber membrane functionalized with tris(hydroxymethyl)aminomethane (P-Tris) was used in affinity membrane chromatography for lysozyme adsorption. The effects of pH and protein concentration on lysozyme adsorption were investigated. Based on Langmuir model, the adsorption capacity of P-Tris nanofiber membrane was estimated to be 345.83 mg/g. For the operation of dynamic membrane chromatography with three-layer P-Tris nanofiber membranes, the optimal operating conditions were at pH 9, 1.0 mL/min of feed flow rate, and 2 mg/mL of feed concentration. Chicken egg white (CEW) was applied as the crude feedstock of lysozyme in the optimized dynamic membrane chromatography. The percent recovery and purification factor of lysozyme obtained from the chromatography were 93.28% and 103.98 folds, respectively. Our findings demonstrated the effectiveness of P-Tris affinity nanofiber membrane for the recovery of lysozyme from complex CEW solution.
    Matched MeSH terms: Chromatography, Affinity/methods*
  10. Development of an immunochromatographic lateral flow dipstick for the detection of Mycobacterium tuberculosis 16 kDa antigen (Mtb-strip)
    Mohd Amiruddin MN, Ang GY, Yu CY, Falero-Diaz G, Otero O, Reyes F, et al.
    J Microbiol Methods, 2020 09;176:106003.
    PMID: 32702386 DOI: 10.1016/j.mimet.2020.106003
    Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis (TB). This contagious disease remains a severe health problem in the world. The disease is transmitted via inhalation of airborne droplets carrying Mtb from TB patients. Early detection of the disease is vital to prevent transmission of the infection to people in close contact with the patients. To date, there is a need of a simple, rapid, sensitive and specific diagnostic test for TB. Previous studies showed the potential of Mtb 16 kDa antigen (Ag16) in TB diagnosis. In this study, lateral flow immunoassay, also called simple strip immunoassay or immunochromatographic test (ICT) for detection of Ag16 was developed (Mtb-strip) and assessed as a potential rapid TB diagnosis method. A monoclonal antibody against Ag16 was optimized as the capturing and detection antibody on the Mtb-strip. Parameters affecting the performance of the Mtb-strip were also optimized before a complete prototype was developed. Analytical sensitivity showed that Mtb-strip was capable to detect as low as 125 ng of purified Ag16. The analytical sensitivity of Mtb-strip suggests its potential usefulness in different clinical applications.
    Matched MeSH terms: Chromatography, Affinity/methods*
  11. Fulltext Combined Optimization of Codon Usage and Glycine Supplementation Enhances the Extracellular Production of a β-Cyclodextrin Glycosyltransferase from Bacillus sp. NR5 UPM in Escherichia coli
    Nik-Pa NIM, Sobri MFM, Abd-Aziz S, Ibrahim MF, Kamal Bahrin E, Mohammed Alitheen NB, et al.
    Int J Mol Sci, 2020 May 30;21(11).
    PMID: 32486212 DOI: 10.3390/ijms21113919
    Two optimization strategies, codon usage modification and glycine supplementation, were adopted to improve the extracellular production of Bacillus sp. NR5 UPM β-cyclodextrin glycosyltransferase (CGT-BS) in recombinant Escherichia coli. Several rare codons were eliminated and replaced with the ones favored by E. coli cells, resulting in an increased codon adaptation index (CAI) from 0.67 to 0.78. The cultivation of the codon modified recombinant E. coli following optimization of glycine supplementation enhanced the secretion of β-CGTase activity up to 2.2-fold at 12 h of cultivation as compared to the control. β-CGTase secreted into the culture medium by the transformant reached 65.524 U/mL at post-induction temperature of 37 °C with addition of 1.2 mM glycine and induced at 2 h of cultivation. A 20.1-fold purity of the recombinant β-CGTase was obtained when purified through a combination of diafiltration and nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. This combined strategy doubled the extracellular β-CGTase production when compared to the single approach, hence offering the potential of enhancing the expression of extracellular enzymes, particularly β-CGTase by the recombinant E. coli.
    Matched MeSH terms: Chromatography, Affinity
  12. Fulltext Effective refolding of a cysteine rich glycoside hydrolase family 19 recombinant chitinase from Streptomyces griseus by reverse dilution and affinity chromatography
    Oyeleye AO, Mohd Yusoff SF, Abd Rahim IN, Leow ATC, Saidi NB, Normi YM
    PLoS One, 2020;15(10):e0241074.
    PMID: 33091044 DOI: 10.1371/journal.pone.0241074
    Conventional refolding methods are associated with low yields due to misfolding and high aggregation rates or very dilute proteins. In this study, we describe the optimization of the conventional methods of reverse dilution and affinity chromatography for obtaining high yields of a cysteine rich recombinant glycoside hydrolase family 19 chitinase from Streptomyces griseus HUT6037 (SgChiC). SgChiC is a potential biocontrol agent and a reference enzyme in the study and development of chitinases for various applications. The overexpression of SgChiC was previously achieved by periplasmic localization from where it was extracted by osmotic shock and then purified by hydroxyapatite column chromatography. In the present study, the successful refolding and recovery of recombinant SgChiC (r-SgChiC) from inclusion bodies (IB) by reverse dilution and column chromatography methods is respectively described. Approximately 8 mg of r-SgChiC was obtained from each method with specific activities of 28 and 52 U/mg respectively. These yields are comparable to that obtained from a 1 L culture volume of the same protein isolated from the periplasmic space of E. coli BL21 (DE3) as described in previous studies. The higher yields obtained are attributed to the successful suppression of aggregation by a stepwise reduction of denaturant from high, to intermediate, and finally to low concentrations. These methods are straight forward, requiring the use of fewer refolding agents compared with previously described refolding methods. They can be applied to the refolding of other cysteine rich proteins expressed as inclusion bodies to obtain high yields of actively folded proteins. This is the first report on the recovery of actively folded SgChiC from inclusion bodies.
    Matched MeSH terms: Chromatography, Affinity
  13. Fulltext Production, purification and evaluation of biodegradation potential of PHB depolymerase of Stenotrophomonas sp. RZS7
    Sayyed RZ, Wani SJ, Alarfaj AA, Syed A, El-Enshasy HA
    PLoS One, 2020;15(1):e0220095.
    PMID: 31910206 DOI: 10.1371/journal.pone.0220095
    There are numerous reports on poly-β-hydroxybutyrate (PHB) depolymerases produced by various microorganisms isolated from various habitats, however, reports on PHB depolymerase production by an isolate from plastic rich sites scares. Although PHB has attracted commercial significance, the inefficient production and recovery methods, inefficient purification of PHB depolymerase and lack of ample knowledge on PHB degradation by PHB depolymerase have hampered its large scale commercialization. Therefore, to ensure the biodegradability of biopolymers, it becomes imperative to study the purification of the biodegrading enzyme system. We report the production, purification, and characterization of extracellular PHB depolymerase from Stenotrophomonas sp. RZS7 isolated from a dumping yard rich in plastic waste. The isolate produced extracellular PHB depolymerase in the mineral salt medium (MSM) at 30°C during 4 days of incubation under shaking. The enzyme was purified by three methods namely ammonium salt precipitation, column chromatography, and solvent purification. Among these purification methods, the enzyme was best purified by column chromatography on the Octyl-Sepharose CL-4B column giving optimum yield (0.7993 Umg-1mL-1). The molecular weight of purified PHB depolymerase was 40 kDa. Studies on the assessment of biodegradation of PHB in liquid culture medium and under natural soil conditions confirmed PHB biodegradation potential of Stenotrophomonas sp. RZS7. The results obtained in Fourier-Transform Infrared (FTIR) analysis, High-Performance Liquid Chromatography (HPLC) study and Gas Chromatography Mass-Spectrometry (GC-MS) analysis confirmed the biodegradation of PHB in liquid medium by Stenotrophomonas sp. RZS7. Changes in surface morphology of PHB film in soil burial as observed in Field Emission Scanning Electron Microscopy (FESEM) analysis confirmed the biodegradation of PHB under natural soil environment. The isolate was capable of degrading PHB and it resulted in 87.74% biodegradation. A higher rate of degradation under the natural soil condition is the result of the activity of soil microbes that complemented the biodegradation of PHB by Stenotrophomonas sp. RZS7.
    Matched MeSH terms: Chromatography, Affinity
  14. Fulltext Expression, purification and characterization of the dimeric protruding domain of Macrobrachium rosenbergii nodavirus capsid protein expressed in Escherichia coli
    Chong LC, Ganesan H, Yong CY, Tan WS, Ho KL
    PLoS One, 2019;14(2):e0211740.
    PMID: 30707739 DOI: 10.1371/journal.pone.0211740
    Macrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which seriously impedes the production of the giant freshwater prawn and has a major economic impact. MrNV contains two segmented RNA molecules, which encode the RNA dependent RNA polymerase (RdRp) and the capsid protein (MrNV-CP) containing 371 amino acid residues. MrNV-CP comprises of the Shell (S) and the Protruding (P) domains, ranging from amino acid residues 1-252 and 253-371, respectively. The P-domain assembles into dimeric protruding spikes, and it is believed to be involved in host cell attachment and internalization. In this study, the recombinant P-domain of MrNV-CP was successfully cloned and expressed in Escherichia coli, purified with an immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) up to ~90% purity. Characterization of the purified recombinant P-domain with SEC revealed that it formed dimers, and dynamic light scattering (DLS) analysis demonstrated that the hydrodynamic diameter of the dimers was ~6 nm. Circular dichroism (CD) analysis showed that the P-domain contained 67.9% of beta-sheets, but without alpha-helical structures. This is in good agreement with the cryo-electron microscopic analysis of MrNV which demonstrated that the P-domain contains only beta-stranded structures. Our findings of this study provide essential information for the production of the P-domain of MrNV-CP that will aid future studies particularly studies that will shed light on anti-viral drug discovery and provide an understanding of virus-host interactions and the viral pathogenicity.
    Matched MeSH terms: Chromatography, Affinity
  15. Fulltext Spray-dried immobilized lipase from Geobacillus sp. strain ARM in sago
    Mohtar NS, Abdul Rahman MB, Mustafa S, Mohamad Ali MS, Raja Abd Rahman RNZ
    PeerJ, 2019;7:e6880.
    PMID: 31183251 DOI: 10.7717/peerj.6880
    Sago starch is traditionally used as food especially in Southeast Asia. Generally, sago is safe for consumption, biodegradable, easily available and inexpensive. Therefore, this research was done to expand the potential of sago by using it as a support for enzyme immobilization. In this study, ARM lipase, which was isolated from Geobacillus sp. strain ARM, was overexpressed in Escherichia coli system and then purified using affinity chromatography. The specific activity of the pure enzyme was 650 U/mg, increased 7 folds from the cell lysate. The purified enzyme was immobilized in gelatinized sago and spray-dried by entrapment technique in order to enhance the enzyme operational stability for handling at high temperature and also for storage. The morphology of the gelatinized sago and immobilized enzyme was studied by scanning electron microscopy. The results showed that the spray-dried gelatinized sago was shrunken and became irregular in structure as compared to untreated sago powder. The surface areas and porosities of spray-dried gelatinized sago with and without the enzyme were analyzed using BET and BJH method and have shown an increase in surface area and decrease in pore size. The immobilized ARM lipase showed good performance at 60-80  °C, with a half-life of 4 h and in a pH range 6-9. The immobilized enzyme could be stored at 10 °C with the half-life for 9 months. Collectively, the spray-dried immobilized lipase shows promising capability for industrial uses, especially in food processing.
    Matched MeSH terms: Chromatography, Affinity
  16. Direct recovery of malate dehydrogenase from highly turbid yeast cell homogenate using dye-ligand affinity chromatography in stirred fluidized bed
    Chen KH, Lee SY, Show PL, Hong SC, Chang YK
    J Chromatogr B Analyt Technol Biomed Life Sci, 2018 Nov 15;1100-1101:65-75.
    PMID: 30292951 DOI: 10.1016/j.jchromb.2018.09.039
    Dye-ligand affinity chromatography in a stirred fluidized bed has been developed for the rapid recovery of malate dehydrogenase (MDH) from highly turbid baker's yeast cell homogenate in a single step. The most suitable dye, namely Reactive Orange 4, in its optimal immobilized concentration of 8.78 mg/mL was immobilized onto high-density STREAMLINE matrix. To further examine optimal adsorption and elution conditions, the enzyme recovery operation was carried out using unclarified cell homogenates in stirred fluidized bed system. Aiming to develop a non-specific eluent, namely NaCl, to effectively elute the MDH adsorbed, direct recovery of MDH from highly turbid cell homogenate (50% w/v) in a stirred fluidized bed adsorption system was performed. The proposed system successfully achieved a recovery yield of 73.6% and a purification factor of 73.5 in a single step by using 0.6 M NaCl as an eluent at a high liquid velocity of 200 cm/h.
    Matched MeSH terms: Chromatography, Affinity/methods*
  17. Fulltext Immunochromatographic test for rapid serological diagnosis of human angiostrongyliasis
    Eamsobhana P, Tungtrongchitr A, Wanachiwanawin D, Yong HS
    Int J Infect Dis, 2018 Aug;73:69-71.
    PMID: 29908250 DOI: 10.1016/j.ijid.2018.06.005
    OBJECTIVES: The serological diagnosis of human infection with Angiostrongylus cantonensis remains problematic because there are no commercially available validated tests. Most laboratories use domestically prepared tests such as the enzyme-linked immunosorbent assay (ELISA) or immunoblotting. Since laboratory facilities are not always available in endemic areas, we developed and assessed a rapid lateral flow immunochromatographic assay (AcQuickDx Test) to detect anti-A. cantonensis antibodies in human serum.

    METHODS: The test device was assembled with purified 31-kDa glycoprotein as diagnostic antigen and with gold-labelled anti-human immunoglublin-G as the detector reagent. A total of 97 serum samples were tested - 19 samples from clinically diagnosed patients with detectable A. cantonensis-specific antibody in immunoblotting; 43 samples from patients with other parasitic diseases, i.e. gnathostomiasis (n=13), toxocariasis (n=2), trichinellosis (n=2), hookworm infection (n=4), filariasis (n=5), cysticercosis (n=9), paragonimiasis (n=2), opisthorchiasis (n=3), and malaria (n=3); and 35 samples from normal healthy subjects.

    RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of AcQuickDx Test to detect anti-A. cantonensis specific antibodies in serologically confirmed angiostrongyliasis cases, were 100%, 98.72%, 95% and 100%, respectively. Positive AcQuickDx was observed in 1 of 4 cases with hookworm infections. No positive AcQuickDx was observed in cases with other parasitic diseases, and the individual healthy subjects.

    CONCLUSIONS: AcQuickDx Test is rapid, highly sensitive and specific, and easy to perform without additional equipment or ancillary supplies. It yields results that are interpreted visually, and possesses a long shelf-life at room temperature. Thus, it can be applied as an additional test for clinical diagnostic support of angiostrongyliasis either in conventional laboratories or for remote areas where laboratory infrastructure is not available.

    Matched MeSH terms: Chromatography, Affinity/methods*
  18. Identification of a target protein of Hydra actinoporin-like toxin-1 (HALT-1) using GST affinity purification and SILAC-based quantitative proteomics
    Ameirika, Sha HX, Hwang JS
    Toxicon, 2017 Jul;133:153-161.
    PMID: 28478056 DOI: 10.1016/j.toxicon.2017.05.007
    Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata. HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein, binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions.
    Matched MeSH terms: Chromatography, Affinity
  19. Fulltext Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein
    Rahaman SN, Mat Yusop J, Mohamed-Hussein ZA, Ho KL, Teh AH, Waterman J, et al.
    Acta Crystallogr F Struct Biol Commun, 2016 Mar;72(Pt 3):207-13.
    PMID: 26919524 DOI: 10.1107/S2053230X16002016
    C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 M magnesium chloride hexahydrate, 0.1 M sodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parameters a = 59.32, b = 65.35, c = 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.
    Matched MeSH terms: Chromatography, Affinity
  20. Expression of a codon-optimised recombinant Ara h 2.02 peanut allergen in Escherichia coli
    Lew MH, Lim RL
    Appl Microbiol Biotechnol, 2016 Jan;100(2):661-71.
    PMID: 26411458 DOI: 10.1007/s00253-015-6953-y
    Current diagnostic tools for peanut allergy using crude peanut extract showed low predictive value and reduced specificity for detection of peanut allergen-specific immunoglobulin E (IgE). The Ara h 2.02, an isoform of the major peanut allergen Ara h 2, contains three IgE epitope recognition sequence of 'DPYSPS' and may be a better reagent for component resolve diagnosis. This research aimed to generate a codon-optimised Ara h 2.02 gene for heterologous expression in Escherichia coli and allergenicity study of this recombinant protein. The codon-optimised gene was generated by PCR using overlapping primers and cloned into the pET-28a (+) expression vector. Moderate expression of a 22.5 kDa 6xhistidine-tagged recombinant Ara h 2.02 protein (6xHis-rAra h 2.02) in BL21 (DE3) host cells was observed upon induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). The insoluble recombinant protein was purified under denaturing condition using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and refolded by dialysis in decreasing urea concentration, amounting to a yield of 74 mg/l of expression culture. Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) and immunoblot analysis confirmed the production of the recombinant 6xHis-rAra h 2.02. The refolded recombinant 6xHis-rAra h 2.02, with or without adjuvant, was able to elicit comparable level of allergen-specific IgE and IgG1 in sensitised Balb/c mice. In addition, the specific IgE antibodies raised against the recombinant protein were able to recognise the native Ara h 2 protein, demonstrating its allergenicity and potential as a reagent for diagnosis and therapeutic study.
    Matched MeSH terms: Chromatography, Affinity
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