Displaying publications 1 - 20 of 56 in total

Abstract:
Sort:
  1. 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
  2. Fulltext MALDI-target integrated platform for affinity-captured protein digestion
    Ahmad-Tajudin A, Adler B, Ekström S, Marko-Varga G, Malm J, Lilja H, et al.
    Anal Chim Acta, 2014 Jan 7;807:1-8.
    PMID: 24356215 DOI: 10.1016/j.aca.2013.08.051
    To address immunocapture of proteins in large cohorts of clinical samples high throughput sample processing is required. Here a method using the proteomic sample platform, ISET (integrated selective enrichment target) that integrates highly specific immunoaffinity capture of protein biomarker, digestion and sample cleanup with a direct interface to mass spectrometry is presented. The robustness of the on-ISET protein digestion protocol was validated by MALDI MS analysis of model proteins, ranging from 40 fmol to 1 pmol per nanovial. On-ISET digestion and MALDI MS/MS analysis of immunoaffinity captured disease-associated biomarker PSA (prostate specific antigen) from human seminal plasma are presented.
    Matched MeSH terms: Chromatography, Affinity
  3. Purification and characterisation of an F16L mutant of a thermostable lipase
    Ali MS, Yun CC, Chor AL, Rahman RN, Basri M, Salleh AB
    Protein J, 2012 Mar;31(3):229-37.
    PMID: 22350313 DOI: 10.1007/s10930-012-9395-8
    A mutant of the lipase from Geobacillus sp. strain T1 with a phenylalanine to leucine substitution at position 16 was overexpressed in Escherichia coli strain BL21(De3)pLysS. The crude enzyme was purified by two-step affinity chromatography with a final recovery and specific activity of 47.4 and 6,315.8 U/mg, respectively. The molecular weight of the purified F16L lipase was approximately 43 kDa by 12% SDS-PAGE analysis. The F16L lipase was demonstrated to be a thermophilic enzyme due its optimum temperature at 70 °C and showed stability over a temperature range of 40-60 °C. The enzyme exhibited an optimum pH 7 in phosphate buffer and was relatively stable at an alkaline pH 8-9. Metal ions such as Ca(2+), Mn(2+), Na(+), and K(+) enhanced the lipase activity, but Mg(2+), Zn(2+), and Fe(2+) inhibited the lipase. All surfactants tested, including Tween 20, 40, 60, 80, Triton X-100, and SDS, significantly inhibited the lipolytic action of the lipase. A high hydrolytic rate was observed on long-chain natural oils and triglycerides, with a notable preference for olive oil (C18:1; natural oil) and triolein (C18:1; triglyceride). The F16L lipase was deduced to be a metalloenzyme because it was strongly inhibited by 5 mM EDTA. Moderate inhibition was observed in the presence of PMSF at a similar concentration, indicating that serine residues are involved in its catalytic action. Further, the activity was not impaired by water-miscible solvents, including methanol, ethanol, and acetone.
    Matched MeSH terms: Chromatography, Affinity
  4. 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
  5. 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
  6. Purification and comparison of intracellular proteinase A in Candida spp. isolates from Malaysian and Iranian patients and infected mice
    Amri Saroukolaei S, Pei Pei C, Shokri H, Asadi F
    J Mycol Med, 2012 Jun;22(2):149-59.
    PMID: 23518017 DOI: 10.1016/j.mycmed.2012.01.002
    To compare the specific intracellular proteinase A activity in clinical isolates of Candida species isolated from Iranian and Malaysian patients, the blood and kidneys of mice infected by Candida cells isolated from these human patients.
    Matched MeSH terms: Chromatography, Affinity
  7. Fulltext Investigation on the conA binding properties of Klebsiella pneumoniae
    Anuar AS, Tay ST
    Trop Biomed, 2014 Dec;31(4):802-12.
    PMID: 25776607 MyJurnal
    Klebsiella pneumoniae is a healthcare-associated bacterial pathogen which causes severe diseases in immunocompromised individuals. Concanavalin A (conA), a lectin which recognizes proteins with mannose or glucose residues, has been reported to agglutinate K. pneumoniae and hence, is postulated to have therapeutical potential for K. pneumoniae-induced liver infection. This study investigated the conA binding properties of a large collection of clinical isolates of K. pneumoniae. ConA agglutination reaction was demonstrated by 94 (51.4%) of 183 K. pneumoniae isolates using a microtiter plate assay. The conA agglutination reactions were inhibited in the presence of 2.5 mg/ml D-mannose and 2.5 mg/ml glucose, and following pretreatment of the bacterial suspension with protease and heating at 80ºC. Majority of the positive isolates originated from respiratory specimens. Isolation of conA-binding proteins from K. pneumoniae ATCC 700603 strain was performed using conA affinity column and the conA binding property of the eluted proteins was confirmed by western blotting analysis using conA-HRP conjugates. Proteins with molecular weights ranging from 35 to 60 kDa were eluted from the conA affinity column, of which four were identified as outer membrane protein precursor A (37 kDa), outer membrane protein precursor C (40 kDa), enolase (45 kDa) and chaperonin (60 kDa) using mass spectrometry analysis. Several conA binding proteins (including 45 and 60 kDa) were found to be immunogenic when reacted with rabbit anti-Klebsiella antibody. The function and interplay of the conA binding proteins in bacterium-host cell relationship merits further investigation.
    Matched MeSH terms: Chromatography, Affinity
  8. 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*
  9. Fulltext Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the histone-like HU protein from Spiroplasma melliferum KC3
    Boyko K, Gorbacheva M, Rakitina T, Korzhenevskiy D, Vanyushkina A, Kamashev D, et al.
    Acta Crystallogr F Struct Biol Commun, 2015 Jan 01;71(Pt 1):24-7.
    PMID: 25615963 DOI: 10.1107/S2053230X14025333
    HU proteins belong to the nucleoid-associated proteins (NAPs) that are involved in vital processes such as DNA compaction and reparation, gene transcription etc. No data are available on the structures of HU proteins from mycoplasmas. To this end, the HU protein from the parasitic mycoplasma Spiroplasma melliferum KC3 was cloned, overexpressed in Escherichia coli and purified to homogeneity. Prismatic crystals of the protein were obtained by the vapour-diffusion technique at 4°C. The crystals diffracted to 1.36 Å resolution (the best resolution ever obtained for a HU protein). The diffraction data were indexed in space group C2 and the structure of the protein was solved by the molecular-replacement method with one monomer per asymmetric unit.
    Matched MeSH terms: Chromatography, Affinity
  10. 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*
  11. 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*
  12. 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*
  13. 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
  14. 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*
  15. 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*
  16. 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
  17. 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
  18. Natural occurrence of ochratoxin A contamination in commercial black and white pepper products
    Jalili M, Jinap S, Radu S
    Mycopathologia, 2010 Oct;170(4):251-8.
    PMID: 20526682 DOI: 10.1007/s11046-010-9320-7
    The concentration of ochratoxin A (OTA) in 120 commercial pepper (84 pre-packed and 36 bulk samples), which consist of local and imported white and black pepper in powder and seed form in Malaysia were determined. The objective of the study was to investigate and compare OTA concentration in black pepper and white pepper being commercialized in Malaysia. Determination method was based on HPLC with fluorescence detection coupled with immunoaffinity column clean-up step. Mobile phase consisted of acetonitrile-water-acetic acid (49.5:49.5:1.0, v/v/v), and flow rate was 1 ml/min. The LOD was 0.02 ng/g, and the average recovery values of OTA ranged from 79.5 to 92.0% in black pepper and 81.2-90.3% in white pepper. A total of 57 samples (47.5%) were contaminated with OTA ranging from 0.15 to 13.58 ng/g. The results showed that there was a significant difference between type of pepper and brands. OTA concentration in black pepper was significantly higher than white pepper (p < 0.05). The highest concentration of ochratoxin, 13.58 ng/g, was detected in a sample of black pepper seed followed by 12.64 ng/g in a sample of black pepper powder, both were bulk samples purchased from open market.
    Matched MeSH terms: Chromatography, Affinity
  19. Fulltext Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis
    Kalyanasundram J, Chia SL, Song AA, Raha AR, Young HA, Yusoff K
    BMC Biotechnol, 2015;15:113.
    PMID: 26715153 DOI: 10.1186/s12896-015-0231-z
    The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus.
    Matched MeSH terms: Chromatography, Affinity
  20. Fulltext Helicobacter pylori recombinant UreG protein: cloning, expression, and assessment of its seroreactivity
    Khalilpour A, Osman S, Yunus MH, Santhanam A, Vellasamy N, Noordin R
    BMC Res Notes, 2014;7:809.
    PMID: 25406411 DOI: 10.1186/1756-0500-7-809
    Helicobacter pylori is a human pathogen and during the process of infection, antigens from the bacterium elicit strong host humoral immune responses. In our previous report, native H. pylori UreG protein showed good reactivity with sera from H. pylori patients. This study was aimed at producing the recombinant form of the protein (rUreG) and determining its seroreactivities.
    Matched MeSH terms: Chromatography, Affinity
Related Terms
Filters
Contact Us

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

External Links