Displaying publications 141 - 160 of 363 in total

Abstract:
Sort:
  1. Cloning and expression of the phosphoprotein gene of Newcastle disease virus in Escherichia coli
    Kho CL, Tan WS, Yusoff K
    J. Biochem. Mol. Biol. Biophys., 2002 Apr;6(2):117-21.
    PMID: 12186767
    The phosphoprotein (P) gene of a heat stable Newcastle disease virus (NDV) was cloned, sequenced and expressed in Escherichia coli. SDS-PAGE analysis of the recombinant P protein (395 amino acids) and a C-terminal extension derivative (424 amino acids), gave rise to two distinct protein bands with molecular masses of approximately 53-55 and 56-58 kDa, respectively, which are approximately 26-30% heavier than those calculated from the deduced amino acid sequences. The differences in molecular mass on SDS-PAGE are thought to be attributed to the acidic nature of the P protein (pI=6.27) and also the different degrees of phosphorylation in the prokaryotic cell. Amino acid sequence comparison of the P protein among the published NDV strains showed that they were highly conserved particularly at the putative phosphorylation sites.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/chemistry
  2. Solubility, immunogenicity and physical properties of the nucleocapsid protein of Nipah virus produced in Escherichia coli
    Tan WS, Ong ST, Eshaghi M, Foo SS, Yusoff K
    J Med Virol, 2004 May;73(1):105-12.
    PMID: 15042656
    The nucleocapsid (N) protein of Nipah virus (NiV) can be produced in three Escherichia coli strains [TOP10, BL21(DE3) and SG935] under the control of trc promoter. However, most of the product existed in the form of insoluble inclusion bodies. There was no improvement in the solubility of the product when this protein was placed under the control of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21(DE3) cell cultures. Solubility analysis of N- and C-terminally deleted mutants revealed that the full-length N protein has the highest solubility. The soluble N protein could be purified efficiently by sucrose gradient centrifugation and nickel affinity chromatography. Electron microscopic analysis of the purified product revealed that the N protein assembled into herringbone-like particles of different lengths. The C-terminal end of the N protein contains the major antigenic region when probed with antisera from humans and pigs infected naturally.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology; Recombinant Proteins/chemistry
  3. Recombinant expression of the larval excretory-secretory antigen TES-120 of Toxocara canis in the methylotrophic yeast Pichia pastoris
    Fong MY, Lau YL
    Parasitol Res, 2004 Jan;92(2):173-6.
    PMID: 14655048
    A gene encoding the larval excretory-secretory antigen TES-120 of the dog ascarid worm Toxocara canis was cloned into the methylotrophic yeast Pichia pastoris. Specificity of the recombinant TES-120 antigen produced by the yeast was investigated. Forty-five human serum samples from patients infected with different()parasitic organisms, including 8 cases of toxocariasis, were tested against the recombinant antigen in immunoblot assays. Results from the assays showed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology*; Recombinant Proteins/metabolism
  4. Biochemical Characterization of the Cytochrome P450 CYP107CB2 from Bacillus lehensis G1
    Ang SS, Salleh AB, Chor LT, Normi YM, Tejo BA, Rahman MBA, et al.
    Protein J, 2018 04;37(2):180-193.
    PMID: 29508210 DOI: 10.1007/s10930-018-9764-z
    The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  5. 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: Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  6. Elucidating endotoxin-biomolecule interactions with FRET: extending the frontiers of their supramolecular complexation
    Obeng EM, Dullah EC, Razak NSA, Danquah MK, Budiman C, Ongkudon CM
    J Biol Methods, 2017;4(2):e71.
    PMID: 31453229 DOI: 10.14440/jbm.2017.172
    Endotoxin has been one of the topical chemical contaminants of major concern to researchers, especially in the field of bioprocessing. This major concern of researchers stems from the fact that the presence of Gram-negative bacterial endotoxin in intracellular products is unavoidable and requires complex downstream purification steps. For instance, endotoxin interacts with recombinant proteins, peptides, antibodies and aptamers and these interactions have formed the foundation for most biosensors for endotoxin detection. It has become imperative for researchers to engineer reliable means/techniques to detect, separate and remove endotoxin, without compromising the quality and quantity of the end-product. However, the underlying mechanism involved during endotoxin-biomolecule interaction is still a gray area. The use of quantitative molecular microscopy that provides high resolution of biomolecules is highly promising, hence, may lead to the development of improved endotoxin detection strategies in biomolecule preparation. Förster resonance energy transfer (FRET) spectroscopy is one of the emerging most powerful tools compatible with most super-resolution techniques for the analysis of molecular interactions. However, the scope of FRET has not been well-exploited in the analysis of endotoxin-biomolecule interaction. This article reviews endotoxin, its pathophysiological consequences and the interaction with biomolecules. Herein, we outline the common potential ways of using FRET to extend the current understanding of endotoxin-biomolecule interaction with the inference that a detailed understanding of the interaction is a prerequisite for the design of strategies for endotoxin identification and removal from protein milieus.
    Matched MeSH terms: Recombinant Proteins
  7. Fulltext Serum from Nipah Virus Patients Recognises Recombinant Viral Proteins Produced in Escherichia coli
    Tiong V, Lam CW, Phoon WH, AbuBakar S, Chang LY
    Jpn J Infect Dis, 2017 Jan 24;70(1):26-31.
    PMID: 27169942 DOI: 10.7883/yoken.JJID.2015.501
    The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology*; Recombinant Proteins/isolation & purification
  8. Fulltext A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB
    Salleh AB, Baharuddin SM, Rahman RNZRA, Leow TC, Basri M, Oslan SN
    Microorganisms, 2020 Nov 06;8(11).
    PMID: 33171893 DOI: 10.3390/microorganisms8111738
    Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter 'ragi' from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 °C and 70 °C) and Hygromycin B (50 μg/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDhα). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDhα, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.
    Matched MeSH terms: Recombinant Proteins
  9. High level expression of Glomerella cingulata cutinase in dense cultures of Pichia pastoris grown under fed-batch conditions
    Seman WM, Bakar SA, Bukhari NA, Gaspar SM, Othman R, Nathan S, et al.
    J Biotechnol, 2014 Aug 20;184:219-28.
    PMID: 24910973 DOI: 10.1016/j.jbiotec.2014.05.034
    A Pichia pastoris transformant carrying the cutinase cDNA of Glomerella cingulata was over-expressed in a 5L bioreactor (2.0L working volume) under fed-batch conditions. Bioreactor experiments rely on varying selected parameters in repeated rounds of optimisation: here these included duration of induction, pH and temperature. Highest cell densities (320gL(-1) wet cell weight) with a cutinase production of 3800mgL(-1) and an activity of 434UmL(-1) were achieved 24h after induction with methanol in basal salt medium (at pH 5 and 28°C). Characterisation of the cutinase showed that it was stable between pH 6 and pH 11, had an optimum pH of 8.0 and retained activity for 30min at 50°C (optimum temperature 25°C).The preferred substrates of G. cingulata cutinase were the medium- to long-chain ρ-nitrophenyl esters of ρ-nitrophenylcaprylate (C8), ρ-nitrophenyllaurate (C12) and ρ-nitrophenylmyristate (C14), with the highest catalytic efficiency, kcat/Km of 7.7±0.7mM(-1)s(-1) for ρ-nitrophenylcaprylate. Microscopic analyses showed that the G. cingulata cutinase was also capable of depolymerising the high molecular weight synthetic polyester, polyethylene terephthalate.
    Matched MeSH terms: Recombinant Proteins/biosynthesis*; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  10. Impact of signal peptide and transmembrane segments on expression and biochemical properties of a lipase from Bacillus sphaericus 205y
    Masomian M, Jasni AS, Rahman RNZRA, Salleh AB, Basri M
    J Biotechnol, 2017 Dec 20;264:51-62.
    PMID: 29107669 DOI: 10.1016/j.jbiotec.2017.10.014
    A total of 97 amino acids, considered as the signal peptide and transmembrane segments were removed from 205y lipase gene using polymerase chain reaction technique that abolished the low activity of this enzyme. The mature enzyme was expressed in Escherichia coli using pBAD expression vector, which gave up to a 13-fold increase in lipase activity. The mature 205y lipase (without signal peptide and transmembrane; -SP/TM) was purified to homogeneity using the isoelectric focusing technique with 53% recovery. Removing of the signal peptide and transmembrane segments had resulted in the shift of optimal pH, an increase in optimal temperature and tolerance towards more water-miscible organic solvents as compared to the characteristics of open reading frame (ORF) of 205y lipase. Also, in the presence of 1mM inhibitors, less decrease in the activity of mature 205y lipase was observed compared to the ORF of the enzyme. Protein structure modeling showed that 205y lipase consisted of an α/β hydrolase fold without lid domain. However, the transmembrane segment could effect on the enzyme activity by covering the active site or aggregation the protein.
    Matched MeSH terms: Recombinant Proteins/genetics*; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  11. Fulltext Construction and heterologous expression of a truncated Haemagglutinin (HA) protein from the avian influenza virus H5N1 in Escherichia coli
    Chee Wei T, Nurul Wahida AG, Shaharum S
    Trop Biomed, 2014 Dec;31(4):792-801.
    PMID: 25776606 MyJurnal
    Malaysia first reported H5N1 poultry case in 2004 and subsequently outbreak in poultry population in 2007. Here, a recombinant gene encoding of peptide epitopes, consisting fragments of HA1, HA2 and a polybasic cleavage site of H5N1 strain Malaysia, was amplified and cloned into pET-47b(+) bacterial expression vector. DNA sequencing and alignment analysis confirmed that the gene had no alteration and in-frame to the vector. Then, His-tagged truncated HA protein was expressed in Escherichia coli BL21 (DE3) under 1 mM IPTG induction. The protein expression was optimized under a time-course induction study and further purified using Ni-NTA agarose under reducing condition. Migration size of protein was detected at 15 kDa by Western blot using anti-His tag monoclonal antibody and demonstrated no discrepancy compared to its calculated molecular weight.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/chemistry
  12. Fulltext Heterologous, Expression, and Characterization of Thermostable Glucoamylase Derived from Aspergillus flavus NSH9 in Pichia pastoris
    Karim KM, Husaini A, Hossain MA, Sing NN, Mohd Sinang F, Hussain MH, et al.
    Biomed Res Int, 2016;2016:5962028.
    PMID: 27504454 DOI: 10.1155/2016/5962028
    A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg(++), Fe(++), Zn(++), Cu(++), and Pb(++)) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries.
    Matched MeSH terms: Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  13. Fulltext Expression and Evaluation of Recombinant Plasmodium knowlesi Merozoite Surface Protein-3 (MSP-3) for Detection of Human Malaria
    De Silva JR, Lau YL, Fong MY
    PLoS One, 2016;11(7):e0158998.
    PMID: 27391270 DOI: 10.1371/journal.pone.0158998
    Malaria remains a major health threat in many parts of the globe and causes high mortality and morbidity with 214 million cases of malaria occurring globally in 2015. Recent studies have outlined potential diagnostic markers and vaccine candidates one of which is the merozoite surface protein (MSP)-3. In this study, novel recombinant Plasmodium knowlesi MSP-3 was cloned, expressed and purified in an Escherichia coli system. Subsequently, the recombinant protein was evaluated for its sensitivity and specificity. The recombinant pkMSP-3 protein reacted with sera from patients with P. knowlesi infection in both Western blot (61%) and ELISA (100%). Specificity-wise, pkMSP-3 did not react with healthy donor sera in either assay and only reacted with a few non-malarial parasitic patient sera in the ELISA assay (3 of 49). In conclusion, sensitivity and specificity of pkMSP-3 was found to be high in the ELISA and Western Blot assay and thus utilising both assays in tandem would provide the best sero-diagnostic result for P. knowlesi infection.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology; Recombinant Proteins/chemistry
  14. Low-factor consumption for major surgery in haemophilia B with long-acting recombinant glycoPEGylated factor IX
    Escobar MA, Tehranchi R, Karim FA, Caliskan U, Chowdary P, Colberg T, et al.
    Haemophilia, 2017 Jan;23(1):67-76.
    PMID: 27480487 DOI: 10.1111/hae.13041
    INTRODUCTION: Surgery in patients with haemophilia B carries a high risk of excessive bleeding and requires adequate haemostatic control until wound healing. Nonacog beta pegol, a long-acting recombinant glycoPEGylated factor IX (FIX), was used in the perioperative management of patients undergoing major surgery.
    AIM: To evaluate the efficacy and safety of nonacog beta pegol in patients with haemophilia B who undergo major surgery.
    METHODS: This was an open-label, multicentre, non-controlled surgery trial aimed at assessing peri- and postoperative efficacy and safety of nonacog beta pegol in 13 previously treated patients with haemophilia B. All patients received a preoperative nonacog beta pegol bolus injection of 80 IU kg-1 . Postoperatively, the patients received fixed nonacog beta pegol doses of 40 IU kg-1 , repeated at the investigator's discretion. Safety assessments included monitoring of immunogenicity and adverse events.
    RESULTS: Intraoperative haemostatic effect was rated 'excellent' or 'good' in all 13 cases. Apart from the preoperative injection, none of the patients needed additional doses of nonacog beta pegol on the day of surgery. The median number of postoperative doses of nonacog beta pegol was 2.0 from days 1 to 6 and 1.5 from days 7 to 13. No unexpected intra- or postoperative complications were observed including deaths or thromboembolic events. No patients developed inhibitors.
    CONCLUSIONS: These results indicated that nonacog beta pegol was safe and effective in the perioperative setting, allowing major surgical interventions in patients with haemophilia B with minimal peri- and postoperative concentrate consumption and infrequent injections as reported with standard FIX products.
    KEYWORDS: Phase III; factor IX; haemophilia B; long-acting recombinant factor IX; nonacog beta pegol; surgery
    Matched MeSH terms: Recombinant Proteins
  15. Virus-like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli
    Kueh CL, Yong CY, Masoomi Dezfooli S, Bhassu S, Tan SG, Tan WS
    Biotechnol Prog, 2017 Mar;33(2):549-557.
    PMID: 27860432 DOI: 10.1002/btpr.2409
    Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme-linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2-12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host-pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549-557, 2017.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  16. Crystallization and X-ray crystallographic analysis of recombinant TylP, a putative γ-butyrolactone receptor protein from Streptomyces fradiae
    Mohd-Sharif N, Shaibullah S, Givajothi V, Tan CS, Ho KL, Teh AH, et al.
    Acta Crystallogr F Struct Biol Commun, 2017 02 01;73(Pt 2):109-115.
    PMID: 28177322 DOI: 10.1107/S2053230X17001212
    TylP is one of five regulatory proteins involved in the regulation of antibiotic (tylosin) production, morphological and physiological differentiation in Streptomyces fradiae. Its function is similar to those of various γ-butyrolactone receptor proteins. In this report, N-terminally His-tagged recombinant TylP protein (rTylP) was overproduced in Escherichia coli and purified to homogeneity. The rTylP protein was crystallized from a reservoir solution comprising 34%(v/v) ethylene glycol and 5%(v/v) glycerol. The protein crystals diffracted X-rays to 3.05 Å resolution and belonged to the trigonal space group P3121, with unit-cell parameters a = b = 126.62, c = 95.63 Å.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  17. Fulltext An evaluation of a recombinant multiepitope based antigen for detection of Toxoplasma gondii specific antibodies
    Hajissa K, Zakaria R, Suppian R, Mohamed Z
    BMC Infect Dis, 2017 12 29;17(1):807.
    PMID: 29284420 DOI: 10.1186/s12879-017-2920-9
    BACKGROUND: The inefficiency of the current tachyzoite antigen-based serological assays for the serodiagnosis of Toxoplasma gondii infection mandates the need for acquirement of reliable and standard diagnostic reagents. Recently, epitope-based antigens have emerged as an alternative diagnostic marker for the achievement of highly sensitive and specific capture antigens. In this study, the diagnostic utility of a recombinant multiepitope antigen (USM.TOXO1) for the serodiagnosis of human toxoplasmosis was evaluated.

    METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis.

    RESULTS: The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody.

    CONCLUSIONS: This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.

    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology; Recombinant Proteins/metabolism
  18. 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: Recombinant Proteins/genetics; Recombinant Proteins/immunology; Recombinant Proteins/metabolism
  19. Self-assembling amyloid-like nanostructures from SARS-CoV-2 S1, S2, RBD and N recombinant proteins
    Morozova OV, Manuvera VA, Barinov NA, Subcheva EN, Laktyushkin VS, Ivanov DA, et al.
    Arch Biochem Biophys, 2024 Feb;752:109843.
    PMID: 38072298 DOI: 10.1016/j.abb.2023.109843
    Self-assembling nanoparticles (saNP) and nanofibers were found in the recombinant coronavirus SARS-CoV-2 S1, S2, RBD and N proteins purified by affinity chromatography using Ni Sepharose. Scanning electron (SEM), atomic force (AFM) microscopy on mica or graphite surface and in liquid as well as dynamic light scattering (DLS) revealed nanostructures of various sizes. AFM in liquid cell without drying on the surface showed mean height of S1 saNP 80.03 nm, polydispersity index (PDI) 0.006; for S2 saNP mean height 93.32 nm, PDI = 0.008; for N saNP mean height 16.71 nm, PDI = 0.99; for RBD saNP mean height 16.25 nm, PDI = 0.55. Ratios between the height and radius of each saNP in the range 0.1-0.5 suggested solid protein NP but not vesicles with internal empty spaces. The solid but not empty structures of the protein saNP were also confirmed by STEM after treatment of saNP with the standard contrasting agent uranyl acetate. The saNP remained stable after multiple freeze-thaw cycles in water and hyperosmotic solutions for 2 years at -20 °C. Receptor-mediated penetration of the SARS-CoV-2 S1 and RBD saNP in the African green mokey kidney Vero cells with the specific receptors for β-coronavirus reproduction was more efficient compared to unspecific endocytosis into MDCK cells without the specific receptors. Amyloid-like structures were revealed in the SARS-CoV-2 S1, S2, RBD and N saNP by means of their interaction with Thioflavin T and Congo Red dyes. Taken together, spontaneous formation of the amyloid-like self-assembling nanostructures due to the internal affinity of the SARS-CoV-2 virion proteins might induce proteinopathy in patients, including conformational neurodegenerative diseases, change stability of vaccines and diagnostic systems.
    Matched MeSH terms: Recombinant Proteins
  20. Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9
    Karim KMR, Husaini A, Sing NN, Tasnim T, Mohd Sinang F, Hussain H, et al.
    Protein Expr Purif, 2019 12;164:105462.
    PMID: 31351992 DOI: 10.1016/j.pep.2019.105462
    The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
Related Terms
Filters
Contact Us

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

External Links