Displaying publications 81 - 100 of 133 in total

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  1. 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: Escherichia coli/metabolism
  2. Purification of recombinant hepatitis B core antigen from unclarified Escherichia coli feedstock using phage-immobilized expanded bed adsorption chromatography
    Ng MY, Tan WS, Tey BT
    J Chromatogr B Analyt Technol Biomed Life Sci, 2012 Aug 15;903:60-7.
    PMID: 22819608 DOI: 10.1016/j.jchromb.2012.06.043
    Fusion M13 phage with disulfide constrained heptapeptide, C-WSFFSNI-C, inserted into the minor coat protein (gpIII), has been selected in the current study as ligand in direct purification of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli (E. coli) feedstock. The selected fusion phage showed strong association with the surface of the core particle. In the present study, this fusion M13 phage was immobilized onto Streamline base matrix via epoxy activation and used as adsorbent to capture HBcAg from crude E. coli homogenate. The maximum binding capacity for the adsorbent was 3.76 mg/mL with equilibrium coefficient of 1.83 mg/mL. Due to the slow uptake rate of HBcAg by M13 phage-immobilized adsorbents, a modified EBAC operation with recirculation of feedstock into the expanded bed has been investigated in this study. The introduction of feedstock recirculation has led to an 18% increase in yield; however, the purity of the eluted product was reduced by 15% compared with typical EBAC operation. The level of antigenicity exhibited by the core particles purified by both EBAC operations employed in the present study was comparable to that purified using sucrose ultracentrifugation.
    Matched MeSH terms: Escherichia coli/metabolism
  3. Spatial assessment of Langat River water quality using chemometrics
    Juahir H, Zain SM, Aris AZ, Yusoff MK, Mokhtar MB
    J Environ Monit, 2010 Jan;12(1):287-95.
    PMID: 20082024 DOI: 10.1039/b907306j
    The present study deals with the assessment of Langat River water quality with some chemometrics approaches such as cluster and discriminant analysis coupled with an artificial neural network (ANN). The data used in this study were collected from seven monitoring stations under the river water quality monitoring program by the Department of Environment (DOE) from 1995 to 2002. Twenty three physico-chemical parameters were involved in this analysis. Cluster analysis successfully clustered the Langat River into three major clusters, namely high, moderate and less pollution regions. Discriminant analysis identified seven of the most significant parameters which contribute to the high variation of Langat River water quality, namely dissolved oxygen, biological oxygen demand, pH, ammoniacal nitrogen, chlorine, E. coli, and coliform. Discriminant analysis also plays an important role as an input selection parameter for an ANN of spatial prediction (pollution regions). The ANN showed better prediction performance in discriminating the regional area with an excellent percentage of correct classification compared to discriminant analysis. Multivariate analysis, coupled with ANN, is proposed, which could help in decision making and problem solving in the local environment.
    Matched MeSH terms: Escherichia coli/metabolism
  4. Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase
    Ismail A, Illias RM
    J Ind Microbiol Biotechnol, 2017 Dec;44(12):1627-1641.
    PMID: 28921081 DOI: 10.1007/s10295-017-1980-6
    The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
    Matched MeSH terms: Escherichia coli/metabolism
  5. Cloning, extracellular expression and characterization of a predominant beta-CGTase from Bacillus sp. G1 in E. coli
    Ong RM, Goh KM, Mahadi NM, Hassan O, Rahman RN, Illias RM
    J Ind Microbiol Biotechnol, 2008 Dec;35(12):1705-14.
    PMID: 18726621 DOI: 10.1007/s10295-008-0462-2
    The cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from Bacillus sp. G1 was successfully isolated and cloned into Escherichia coli. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,109 bp and encoded a 674 amino acid protein. Purified CGTase exhibited a molecular weight of 75 kDa and had optimum activity at pH 6 and 60 degrees C. Heterologous recombinant protein expression in E. coli is commonly problematic causing intracellular localization and formation of inactive inclusion bodies. This paper shows that the majority of CGTase was secreted into the medium due to the signal peptide of Bacillus sp. G1 that also works well in E. coli, leading to easier purification steps. When reacted with starch, CGTase G1 produced 90% beta-cyclodextrin (CD) and 10% gamma-CD. This enzyme also preferred the economical tapioca starch as a substrate, based on kinetics studies. Therefore, CGTase G1 could potentially serve as an industrial enzyme for the production of beta-CD.
    Matched MeSH terms: Escherichia coli/metabolism
  6. Cloning and characterization of two new thermostable and alkalitolerant α-amylases from the Anoxybacillus species that produce high levels of maltose
    Chai YY, Rahman RN, Illias RM, Goh KM
    J Ind Microbiol Biotechnol, 2012 May;39(5):731-41.
    PMID: 22246222 DOI: 10.1007/s10295-011-1074-9
    Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD-Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6-10. At 60°C in the absence of Ca²⁺, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca²⁺ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.
    Matched MeSH terms: Escherichia coli/metabolism
  7. Comparison of the estimation capabilities of response surface methodology and artificial neural network for the optimization of recombinant lipase production by E. coli BL21
    Nelofer R, Ramanan RN, Rahman RN, Basri M, Ariff AB
    J Ind Microbiol Biotechnol, 2012 Feb;39(2):243-54.
    PMID: 21833714 DOI: 10.1007/s10295-011-1019-3
    Response surface methodology (RSM) and artificial neural network (ANN) were used to optimize the effect of four independent variables, viz. glucose, sodium chloride (NaCl), temperature and induction time, on lipase production by a recombinant Escherichia coli BL21. The optimization and prediction capabilities of RSM and ANN were then compared. RSM predicted the dependent variable with a good coefficient of correlation determination (R² and adjusted R² values for the model. Although the R (2) value showed a good fit, absolute average deviation (AAD) and root mean square error (RMSE) values did not support the accuracy of the model and this was due to the inferiority in predicting the values towards the edges of the design points. On the other hand, ANN-predicted values were closer to the observed values with better R², adjusted R², AAD and RMSE values and this was due to the capability of predicting the values throughout the selected range of the design points. Similar to RSM, ANN could also be used to rank the effect of variables. However, ANN could not predict the interactive effect between the variables as performed by RSM. The optimum levels for glucose, NaCl, temperature and induction time predicted by RSM are 32 g/L, 5 g/L, 32°C and 2.12 h, and those by ANN are 25 g/L, 3 g/L, 30°C and 2 h, respectively. The ANN-predicted optimal levels gave higher lipase activity (55.8 IU/mL) as compared to RSM-predicted levels (50.2 IU/mL) and the predicted lipase activity was also closer to the observed data at these levels, suggesting that ANN is a better optimization method than RSM for lipase production by the recombinant strain.
    Matched MeSH terms: Escherichia coli/metabolism*
  8. An effective extracellular protein secretion by an ABC transporter system in Escherichia coli: statistical modeling and optimization of cyclodextrin glucanotransferase secretory production
    Low KO, Mahadi NM, Rahim RA, Rabu A, Abu Bakar FD, Murad AM, et al.
    J Ind Microbiol Biotechnol, 2011 Sep;38(9):1587-97.
    PMID: 21336875 DOI: 10.1007/s10295-011-0949-0
    Direct transport of recombinant protein from cytosol to extracellular medium offers great advantages, such as high specific activity and a simple purification step. This work presents an investigation on the potential of an ABC (ATP-binding cassette) transporter system, the hemolysin transport system, for efficient protein secretion in Escherichia coli (E. coli). A higher secretory production of recombinant cyclodextrin glucanotransferase (CGTase) was achieved by a new plasmid design and subsequently by optimization of culture conditions via central composite design. An improvement of at least fourfold extracellular recombinant CGTase was obtained using the new plasmid design. The optimization process consisted of 20 experiments involving six star points and six replicates at the central point. The predicted optimum culture conditions for maximum recombinant CGTase secretion were found to be 25.76 μM IPTG, 1.0% (w/v) arabinose and 34.7°C post-induction temperature, with a predicted extracellular CGTase activity of 68.76 U/ml. Validation of the model gave an extracellular CGTase activity of 69.15 ± 0.71 U/ml, resulting in a 3.45-fold increase compared to the initial conditions. This corresponded to an extracellular CGTase yield of about 0.58 mg/l. We showed that a synergistic balance of transported protein and secretory pathway is important for efficient protein transport. In addition, we also demonstrated the first successful removal of the C-terminal secretion signal from the transported fusion protein by thrombin proteolytic cleavage.
    Matched MeSH terms: Escherichia coli/metabolism*
  9. Topoisomerase I inhibition and DNA cleavage by zinc, copper, and nickel derivatives of 2-[2-bromoethyliminomethyl]-4-[ethoxymethyl]phenol complexes exhibiting anti-proliferation and anti-metastasis activity
    Lee SK, Tan KW, Ng SW
    J Inorg Biochem, 2016 06;159:14-21.
    PMID: 26901628 DOI: 10.1016/j.jinorgbio.2016.02.010
    Three transition metal derivatives (Zn, Cu, and Ni) of 2-[2-bromoethyliminomethyl]-4-[ethoxymethyl]phenol (L) were synthesized by the reaction of the metal salts with the Schiff base ligand in one pot. In the crystal structure of [Zn(L)Br], the Schiff base ligand binds to the metal center through its phenolate oxygen and imine nitrogen, and adopts a distorted tetrahedral geometry. These compounds were found to inhibit topoisomerase I (topo I) activity, induce DNA cleavage and show DNA binding activity. Moreover, these compounds were found to be cytotoxic towards several cancer cell lines (A2780, MCF-7, HT29, HepG2, A549, PC3, LNCaP) and prevent metastasis of PC3. Collectively, Cu(II) complex 2 shows superior activity relative to its Zn(II) and Ni(II) analogs.
    Matched MeSH terms: Escherichia coli/metabolism*
  10. Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates
    Tan YP, Ling TC, Yusoff K, Tan WS, Tey BT
    J Microbiol, 2005 Jun;43(3):295-300.
    PMID: 15995649
    In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.
    Matched MeSH terms: Escherichia coli/metabolism*
  11. Construction and characterization of a Burkholderia pseudomallei wzm deletion mutant
    Yuen CW, Ong EB, Mohamad S, Manaf UA, Najimudin N
    J Microbiol Biotechnol, 2012 Oct;22(10):1336-42.
    PMID: 23075783
    In Burkholderia pseudomallei, the pathogen that causes melioidosis, the gene cluster encoding the capsular polysaccharide, is located on chromosome 1. Among the 19 capsular genes in this cluster, wzm has not been thoroughly studied. To study the function of wzm, we generated a deletion mutant and compared it with the wild-type strain. The mutant produced less biofilm in minimal media and was more sensitive to desiccation and oxidative stress compared with the wild-type strain, indicating that wzm is involved in biofilm formation and membrane integrity. Scanning electron microscopy showed that the bacterial cells of the mutant strain have more defined surfaces with indentations, whereas cells of the wild-type strain do not.
    Matched MeSH terms: Escherichia coli/metabolism
  12. Simultaneous saccharification and fermentation of ground corn stover for the production of fuel ethanol using Phanerochaete chrysosporium, Gloeophyllum trabeum, Saccharomyces cerevisiae, and Escherichia coli K011
    Vincent M, Pometto AL, van Leeuwen JH
    J Microbiol Biotechnol, 2011 Jul;21(7):703-10.
    PMID: 21791956
    Enzymatic saccharification of corn stover using Phanerochaete chrysosporium and Gloeophyllum trabeum and subsequent fermentation of the saccharification products to ethanol by Saccharomyces cerevisiae and Escherichia coli K011 were achieved. Prior to simultaneous saccharification and fermentation (SSF) for ethanol production, solid-state fermentation was performed for four days on ground corn stover using either P. chrysosporium or G. trabeum to induce in situ cellulase production. During SSF with S. cerevisiae or E. coli, ethanol production was the highest on day 4 for all samples. For corn stover treated with P. chrysosporium, the conversion to ethanol was 2.29 g/100 g corn stover with S. cerevisiae as the fermenting organism, whereas for the sample inoculated with E. coli K011, the ethanol production was 4.14 g/100 g corn stover. Corn stover treated with G. trabeum showed a conversion 1.90 and 4.79 g/100 g corn stover with S. cerevisiae and E. coli K011 as the fermenting organisms, respectively. Other fermentation co-products, such as acetic acid and lactic acid, were also monitored. Acetic acid production ranged between 0.45 and 0.78 g/100 g corn stover, while no lactic acid production was detected throughout the 5 days of SSF. The results of our experiment suggest that it is possible to perform SSF of corn stover using P. chrysosporium, G. trabeum, S. cerevisiae and E. coli K011 for the production of fuel ethanol.
    Matched MeSH terms: Escherichia coli/metabolism*
  13. Application of the linear interaction energy method (LIE) to estimate the binding free energy values of Escherichia coli wild-type and mutant arginine repressor C-terminal domain (ArgRc)-l-arginine and ArgRc-l-citrulline protein-ligand complexes
    Asi AM, Rahman NA, Merican AF
    J Mol Graph Model, 2004 Mar;22(4):249-62.
    PMID: 15177077
    Protein-ligand binding free energy values of wild-type and mutant C-terminal domain of Escherichia coli arginine repressor (ArgRc) protein systems bound to L-arginine or L-citrulline molecules were calculated using the linear interaction energy (LIE) method by molecular dynamics (MD) simulation. The binding behaviour predicted by the dissociation constant (K(d)) calculations from the binding free energy values showed preferences for binding of L-arginine to the wild-type ArgRc but not to the mutant ArgRc(D128N). On the other hand, L-citrulline do not favour binding to wild-type ArgRc but prefer binding to mutant ArgRc(D128N). The dissociation constant for the wild-type ArgRc-L-arginine complex obtained in this study is in agreement with reported experimental results. Our results also support the experimental data for the binding of L-citrulline to the mutant ArgRc(D128N). These showed that LIE method for protein-ligand binding free energy calculation could be applied to the wild-type and the mutant E. coli ArgRc-L-arginine and ArgRc-L-citrulline protein-ligand complexes and possibly to other transcriptional repressor-co-repressor systems as well.
    Matched MeSH terms: Escherichia coli/metabolism
  14. Fulltext Optimization of a heterologous signal peptide by site-directed mutagenesis for improved secretion of recombinant proteins in Escherichia coli
    Jonet MA, Mahadi NM, Murad AM, Rabu A, Bakar FD, Rahim RA, et al.
    J. Mol. Microbiol. Biotechnol., 2012;22(1):48-58.
    PMID: 22456489 DOI: 10.1159/000336524
    A heterologous signal peptide (SP) from Bacillus sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase (CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli. Eight mutant SPs were constructed using site-directed mutagenesis to improve the secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an isoleucine residue in the h-region to glycine created a helix-breaking or G-turn motif with decreased hydrophobicity. The mutant SP resulted in 110 and 94% increases in periplasmic and extracellular recombinant CGTase, respectively, compared to the wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrylamyde gel electrophoresis, when this mutated SP was used. The addition of as low as 0.08% glycine at the beginning of cell growth improved cell viability of the E. coli host. Secretory production of other proteins, such as mannosidase, also showed similar improvement, as demonstrated by CGTase production, suggesting that the combination of an optimized SP and a suitable chemical additive leads to significant improvements of extracellular recombinant protein production and cell viability. These findings will be valuable for the extracellular production of recombinant proteins in E. coli.
    Matched MeSH terms: Escherichia coli/metabolism*
  15. 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: Escherichia coli/metabolism
  16. Heat treatment of unclarified Escherichia coli homogenate improved the recovery efficiency of recombinant hepatitis B core antigen
    Ng MY, Tan WS, Abdullah N, Ling TC, Tey BT
    J Virol Methods, 2006 Oct;137(1):134-9.
    PMID: 16860402
    Heat precipitation procedure has been regularly incorporated as a selective purification step in various thermostable proteins expressed in different hosts. This method is efficient in precipitation of most of the host proteins and also deactivates various host proteases that can be harmful to the desired gene products. In this study, introduction of heat treatment procedure in the purification of hepatitis B core antigen (HBcAg) produced in Escherichia coli has been investigated. Thermal treatment of the cell homogenate at 60 degrees C for 30 min prior to subsequent clarification steps has resulted in 1.4 times and 18% higher in purity and recovery yield, respectively, compared to the non-heat-treated cell homogenate. In direct capture of HBcAg by using anion-exchangers from unclarified feedstock, pre-conditioning the feedstock by heat treatment at 60 degrees C for 45 min has increased the recovery yield of HBcAg by 2.9-fold and 42% in purity compared to that treated for 10 min. Enzyme-linked immunosorbent assay (ELISA) analysis showed that the antigenicity of the core particles was not affected by the heat treatment process.
    Matched MeSH terms: Escherichia coli/metabolism*
  17. Production of the matrix protein of Nipah virus in Escherichia coli: virus-like particles and possible application for diagnosis
    Subramanian SK, Tey BT, Hamid M, Tan WS
    J Virol Methods, 2009 Dec;162(1-2):179-83.
    PMID: 19666056 DOI: 10.1016/j.jviromet.2009.07.034
    The broad species tropism of Nipah virus (NiV) coupled with its high pathogenicity demand a rapid search for a new biomarker candidate for diagnosis. The matrix (M) protein was expressed in Escherichia coli and purified using a Ni-NTA affinity column chromatography and sucrose density gradient centrifugation. The recombinant M protein with the molecular mass (Mr) of about 43 kDa was detected by anti-NiV serum and anti-myc antibody. About 50% of the M protein was found to be soluble and localized in cytoplasm when the cells were grown at 30 degrees C. Electron microscopic analysis showed that the purified M protein assembled into spherical particles of different sizes with diameters ranging from 20 to 50 nm. The purified M protein showed significant reactivity with the swine sera collected during the NiV outbreak, demonstrating its potential as a diagnostic reagent.
    Matched MeSH terms: Escherichia coli/metabolism*
  18. Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis
    Raha AR, Chang LY, Sipat A, Yusoff K, Haryanti T
    Lett Appl Microbiol, 2006 Mar;42(3):210-4.
    PMID: 16478506
    The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus.
    Matched MeSH terms: Escherichia coli/metabolism
  19. Fulltext Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli
    Norsyahida A, Rahmah N, Ahmad RM
    Lett Appl Microbiol, 2009 Nov;49(5):544-50.
    PMID: 19832937 DOI: 10.1111/j.1472-765X.2009.02694.x
    To investigate the effects of feeding and induction strategies on the production of BmR1 recombinant antigen.
    Matched MeSH terms: Escherichia coli/metabolism*
  20. Fulltext Isolation and characterization of malaria PfHRP2 specific VNAR antibody fragments from immunized shark phage display library
    Leow CH, Fischer K, Leow CY, Braet K, Cheng Q, McCarthy J
    Malar J, 2018 Oct 24;17(1):383.
    PMID: 30355309 DOI: 10.1186/s12936-018-2531-y
    BACKGROUND: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability.

    METHODS: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken.

    RESULTS: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1-13.

    CONCLUSIONS: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs.

    Matched MeSH terms: Escherichia coli/metabolism
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