Displaying publications 1 - 20 of 22 in total

Abstract:
Sort:
  1. 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
  2. Optimization of conditions for the single step IMAC purification of miraculin from Synsepalum dulcificum
    He Z, Tan JS, Lai OM, Ariff AB
    Food Chem, 2015 Aug 15;181:19-24.
    PMID: 25794715 DOI: 10.1016/j.foodchem.2014.11.166
    In this study, the methods for extraction and purification of miraculin from Synsepalum dulcificum were investigated. For extraction, the effect of different extraction buffers (phosphate buffer saline, Tris-HCl and NaCl) on the extraction efficiency of total protein was evaluated. Immobilized metal ion affinity chromatography (IMAC) with nickel-NTA was used for the purification of the extracted protein, where the influence of binding buffer pH, crude extract pH and imidazole concentration in elution buffer upon the purification performance was explored. The total amount of protein extracted from miracle fruit was found to be 4 times higher using 0.5M NaCl as compared to Tris-HCl and phosphate buffer saline. On the other hand, the use of Tris-HCl as binding buffer gave higher purification performance than sodium phosphate and citrate-phosphate buffers in IMAC system. The optimum purification condition of miraculin using IMAC was achieved with crude extract at pH 7, Tris-HCl binding buffer at pH 7 and the use of 300 mM imidazole as elution buffer, which gave the overall yield of 80.3% and purity of 97.5%. IMAC with nickel-NTA was successfully used as a single step process for the purification of miraculin from crude extract of S. dulcificum.
    Matched MeSH terms: Chromatography, Affinity/methods
  3. 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
  4. 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*
  5. 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*
  6. 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
  7. Fulltext Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases
    Chee CS, Tan IK, Alias Z
    ScientificWorldJournal, 2014;2014:750317.
    PMID: 24892084 DOI: 10.1155/2014/750317
    Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively.
    Matched MeSH terms: Chromatography, Affinity/methods*
  8. Application of champedak mannose-binding lectin in the glycoproteomic profiling of serum samples unmasks reduced expression of alpha-2 macroglobulin and complement factor B in patients with nasopharyngeal carcinoma
    Seriramalu R, Pang WW, Jayapalan JJ, Mohamed E, Abdul-Rahman PS, Bustam AZ, et al.
    Electrophoresis, 2010 Jul;31(14):2388-95.
    PMID: 20575108 DOI: 10.1002/elps.201000164
    The use of lectin affinity chromatography prior to 2-DE separation forms an alternative method to unmask the expression of targeted glycoproteins of lower abundance in serum samples. Reduced expression of alpha-2 macroglobulin (AMG) and complement factor B (CFB) was detected in sera of patients with nasopharyngeal carcinoma (NPC) when pooled serum samples of the patients and those of healthy individuals were subjected to affinity isolation using immobilized champedak mannose-binding lectin and analyzed by 2-DE and densitometry. The AMG and CFB spots were not detected in the 2-DE protein profiles when the same pooled serum samples were subjected to albumin and IgG depletion and neither were they detected when the depleted samples were analyzed by western blotting and lectin detection. Together with other acute-phase response proteins that were previously reported to be altered in expression in NPC patients, AMG and CFB may serve as useful complementary biomarkers for NPC.
    Matched MeSH terms: Chromatography, Affinity/methods*
  9. Evaluation of a method to determine the natural occurrence of aflatoxins in commercial traditional herbal medicines from Malaysia and Indonesia
    Ali N, Hashim NH, Saad B, Safan K, Nakajima M, Yoshizawa T
    Food Chem Toxicol, 2005 Dec;43(12):1763-72.
    PMID: 16019122
    Traditional herbal medicines, popularly known as 'jamu' and 'makjun' in Malaysia and Indonesia, are consumed regularly to promote health. In consideration of their frequent and prolonged consumption, the natural occurrence of aflatoxins (AF) in these products was determined using immunoaffinity column clean-up and high-performance liquid chromatography with pre-column derivatization. The evaluated method, which entails dilution of sample extracts with Tween 20-phosphate buffered saline (1:9, v/v) and a chromatographic system using isocratic mobile phase composed of water-methanol-acetonitrile (70:20:10, v/v/v), was effective in separating AFB1, AFG1 and AFG2 from interference at their retention times. Results were confirmed using post-column derivatization with photochemical reactor. For 23 commercial samples analyzed, mean levels (incidence) of AFB(1), AFB(2) and AFG1 in positive samples were 0.26 (70%), 0.07 (61%) and 0.10 (30%) microg/kg, respectively; one sample was positive for AFG2 at a level of 0.03 (4%) microg/kg. In contrast to the high levels of AF in crude herbal drugs and medicinal plants reported previously by other researchers, the low contamination levels reported in this study may be attributed to the higher selectivity to AF of the method applied. Based on the AFB1 levels and the daily consumption of positive samples, a mean probable daily intake of 0.022 ng/kg body weight was calculated.
    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. 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*
  12. Immunoaffinity extraction and tandem mass spectrometric analysis of human chorionic gonadotropin in doping analysis
    Gam LH, Tham SY, Latiff A
    J Chromatogr B Analyt Technol Biomed Life Sci, 2003 Jul 25;792(2):187-96.
    PMID: 12860026
    A confirmatory and quantitative HPLC-tandem mass spectrometry (MS-MS) method for human chorionic gonadotropin hormone (hCG) at concentrations as low as 5 IU/l following immunoaffinity extraction of the glycoprotein from urine was developed. The extraction method involved retention of urinary hCG in the immunoaffinity column via specific antigen-antibody interaction. A variety of eluents were then used to quantitatively elute hCG from the immunoaffinity column. Qualitative and quantitative analysis of hCG were undertaken using MS-MS by identifying the amino acid sequence of the marker peptide betaT5 obtained from hCG by tryptic digestion and the peak areas of three product ions b(6)(+), b(9)(+) and y(11)(+), respectively.
    Matched MeSH terms: Chromatography, Affinity/methods*
  13. Purification of BmR1 recombinant protein
    Arifin N, Basuni M, Lan CA, Yahya AR, Noordin R
    Protein J, 2010 Oct;29(7):509-15.
    PMID: 20845068 DOI: 10.1007/s10930-010-9281-1
    This paper describes a refinement in the purification step that facilitated the downstream recovery of high purity BmR1 recombinant protein, which is a protein used as a test reagent in the commercialized rapid tests for detection of lymphac filariasis i.e. Brugia Rapid™ and panLF rapid™. Purification was performed by immobilized metal affinity chromatography (IMAC), followed by ion exchange chromatography (IEX). Results showed that a total of 10.27 mg of BmR1 was obtained when IMAC was performed using 20 mM of imidazole and 5 column volume of wash buffer containing 500 mM of NaCl. Purity of the target protein was enhanced when buffer at pH 5.8 was used during the IEX. Two proteins that recurrently appeared below the BmR1 recombinant protein were identified by mass-spectrometry analysis as the same protein, thus they were probably degradation products of BmR1. These strategies improve purity of the target protein to be used in applications such as production of aptamers and monoclonal antibodies.
    Matched MeSH terms: Chromatography, Affinity/methods*
  14. Sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particles
    Opitz L, Lehmann S, Reichl U, Wolff MW
    Biotechnol Bioeng, 2009 Aug 15;103(6):1144-54.
    PMID: 19449393 DOI: 10.1002/bit.22345
    Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture-based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture-derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell-derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM-adsorbers were compared directly to column-based Cellufine sulfate and commercially available cation-exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM-capture step resulted in a higher reduction of dsDNA compared to the tested cation-exchange membrane adsorbers. The productivity of the SCM-based unit operation could be significantly improved by a 30-fold increase in volumetric flow rate during adsorption compared to the bead-based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell-derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM-adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine sulfate.
    Matched MeSH terms: Chromatography, Affinity/methods*
  15. Removal of poly-histidine fusion tags from recombinant proteins purified by expanded bed adsorption
    Abdullah N, Chase HA
    Biotechnol Bioeng, 2005 Nov 20;92(4):501-13.
    PMID: 16080185
    Enzymatic methods have been used to cleave the C- or N-terminus polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.
    Matched MeSH terms: Chromatography, Affinity/methods
  16. 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*
  17. 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*
  18. Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography
    Chong FC, Tan WS, Biak DR, Ling TC, Tey BT
    J Chromatogr B Analyt Technol Biomed Life Sci, 2009 May 15;877(14-15):1561-7.
    PMID: 19395325 DOI: 10.1016/j.jchromb.2009.03.048
    Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.
    Matched MeSH terms: Chromatography, Affinity/methods*
  19. Purification of His-tagged hepatitis B core antigen from unclarified bacterial homogenate using immobilized metal affinity-expanded bed adsorption chromatography
    Yap WB, Tey BT, Alitheen NB, Tan WS
    J Chromatogr A, 2010 May 21;1217(21):3473-80.
    PMID: 20388569 DOI: 10.1016/j.chroma.2010.03.012
    Hepatitis B core antigen (HBcAg) is used as a diagnostic reagent for the detection of hepatitis B virus infection. In this study, immobilized metal affinity-expanded bed adsorption chromatography (IMA-EBAC) was employed to purify N-terminally His-tagged HBcAg from unclarified bacterial homogenate. Streamline Chelating was used as the adsorbent and the batch adsorption experiment showed that the optimal binding pH of His-tagged HBcAg was 8.0 with a binding capacity of 1.8 mg per ml of adsorbent. The optimal elution condition for the elution of His-tagged HBcAg from the adsorbent was at pH 7 in the presence of 500 mM imidazole and 1.5 M NaCl. The IMA-EBAC has successfully recovered 56% of His-tagged HBcAg from the unclarified E. coli homogenate with a purification factor of 3.64. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of the recovered His-tagged HBcAg was not affected throughout the IMA-EBAC purification process and electron microscopy revealed that the protein assembled into virus-like particles (VLP).
    Matched MeSH terms: Chromatography, Affinity/methods*
  20. Fulltext Cloning and expression of Toxoplasma gondii dense granular protein 4 (GRA4) in Pichia pastoris
    Lau YL, Hasan MT, Thiruvengadam G, Idris MM, Init I
    Trop Biomed, 2010 Dec;27(3):525-33.
    PMID: 21399595
    GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
    Matched MeSH terms: Chromatography, Affinity/methods
Related Terms
Filters
Contact Us

Please provide feedback to Administrator (afdal@afpm.org.my)

External Links