Displaying publications 1 - 20 of 362 in total

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  1. Wide dynamic range of surface-plasmon-resonance-based assay for hepatitis B surface antigen antibody optimal detection in comparison with ELISA
    Tam YJ, Zeenathul NA, Rezaei MA, Mustafa NH, Azmi MLM, Bahaman AR, et al.
    Biotechnol Appl Biochem, 2017 Sep;64(5):735-744.
    PMID: 27506960 DOI: 10.1002/bab.1528
    Limit of detection (LOD), limit of quantification, and the dynamic range of detection of hepatitis B surface antigen antibody (anti-HBs) using a surface plasmon resonance (SPR) chip-based approach with Pichia pastoris-derived recombinant hepatitis B surface antigen (HBsAg) as recognition element were established through the scouting for optimal conditions for the improvement of immobilization efficiency and in the use of optimal regeneration buffer. Recombinant HBsAg was immobilized onto the sensor surface of a CM5 chip at a concentration of 150 mg/L in sodium acetate buffer at pH 4 with added 0.6% Triton X-100. A regeneration solution of 20 mM HCl was optimally found to effectively unbind analytes from the ligand, thus allowing for multiple screening cycles. A dynamic range of detection of ∼0.00098-0.25 mg/L was obtained, and a sevenfold higher LOD, as well as a twofold increase in coefficient of variance of the replicated results, was shown as compared with enzyme-linked immunosorbent assay (ELISA). Evaluation of the assay for specificity showed no cross-reactivity with other antibodies tested. The ability of SPR chip-based assay and ELISA to detect anti-HBs in human serum was comparable, indicating that the SPR chip-based assay with its multiple screening capacity has greater advantage over ELISA.
    Matched MeSH terms: Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  2. Virus-like particles of Macrobrachium rosenbergii nodavirus produced in bacteria
    Goh ZH, Tan SG, Bhassu S, Tan WS
    J Virol Methods, 2011 Jul;175(1):74-9.
    PMID: 21536072 DOI: 10.1016/j.jviromet.2011.04.021
    Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism
  3. 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
  4. Fulltext Variant of Helicobacter pylori CagA proteins induce different magnitude of morphological changes in gastric epithelial cells
    Alfizah H, Ramelah M
    Malays J Pathol, 2012 Jun;34(1):29-34.
    PMID: 22870595 MyJurnal
    Infection with Helicobacter pylori cagA-positive strains is associated with gastroduodenal diseases. The CagA protein is injected into gastric epithelial cells and supposedly induces morphological changes termed the 'hummingbird phenotype', which is associated with scattering and increased cell motility. The molecular mechanisms leading to the CagA-dependent morphological changes are only partially known. The present study was carried out to investigate the effect of CagA variants on the magnitude of gastric epithelial cell morphological changes. Recombinant 3' terminal domains of cagA were cloned and expressed in a gastric epithelial cell line and the hummingbird phenotype was quantified by microscopy. The 3' region of the cagA gene of Malaysian H. pylori isolates showed six sub-genotypes that differed in the structural organization of the EPIYA repeat sequences. The percentage of hummingbird cells induced by CagA increased with duration of transfection. The hummingbird phenotype was observed to be more pronounced when CagA with 4 EPIYA motifs rather than 3 or 2 EPIYA motifs was produced. The activity of different CagA variants in the induction of the hummingbird phenotype in gastric epithelial cells depends at least in part on EPIYA motif variability. The difference in CagA genotypes might influence the potential of individual CagAs to cause morphological changes in host cells. Depending on the relative exposure of cells to CagA genotypes, this may contribute to the various disease outcomes caused by H. pylori infection in different individuals.
    Matched MeSH terms: Recombinant Proteins
  5. Validation of a Burkholderia pseudomallei hypothetical protein and determination of its translational start codon using chromosomal integration of His-Tag coding sequence
    Yam H, Abdul Rahim A, Gim Luan O, Samian R, Abdul Manaf U, Mohamad S, et al.
    Protein J, 2012 Mar;31(3):246-9.
    PMID: 22354666 DOI: 10.1007/s10930-012-9398-5
    In this post genomic era, there are a great number of in silico annotated hypothetical genes. However, experimental validation of the functionality of these genes remains tentative. Two of the major challenges faced by researcher are whether these hypothetical genes are protein-coding genes and whether their corresponding predicted translational start codons are correct. In this report, we demonstrate a convenient procedure to validate the presence of a hypothetical gene product of BPSS1356 from Burkholderia pseudomallei as well as its start codon. It was done by integration of a His-Tag coding sequence into C-terminal end of BPSS1356 gene via homologous recombination. We then purified the native protein using affinity chromatography. The genuine start codon of BPSS1356 was then determined by protein N-terminal sequencing.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism
  6. Utility of recombinant LipL41 based IgM and IgG ELISA in diagnosis of canine leptospirosis in an endemic area - a study from Kerala, India
    Ambily R, Mini M, Siju J, Vamshikrishna S, Abhinay G, Gleeja VL, et al.
    Trop Biomed, 2019 Sep 01;36(3):654-663.
    PMID: 33597487
    A study was undertaken to evaluate the relevance of detecting IgM and IgG antibodies in diagnosis of canine leptospirosis in Kerala, a southern state of India, which is endemic for the disease. A total of 205 blood (35 from healthy vaccinated, 30 from healthy unvaccinated and 140 from diseased dogs) and 151 urine samples (11 from healthy vaccinated and 140 from diseased dogs) were collected from three districts of Kerala, Thrissur, Palakkad and Kozhikode with high incidence of leptospirosis. Recombinant LipL41 protein was used as antigen and IgG and IgM based ELISAs were standardized. The results were compared with the gold standard test, microscopic agglutination test (MAT). The MAT positive samples (146 samples) were divided into those having titre >1:800 and those between 1:100 and 1:400 in view that the former constituted the acute cases. It was found that IgM ELISA was more specific and sensitive in detecting acute cases (MAT >1:800) whereas IgG ELISA was less specific. In case of seroprevalence studies (MAT titre 1:100 to 1: 400), IgG ELISA was found to be more sensitive and specific than IgM ELISA. Receiver operating characteristic curves when plotted, revealed the accuracy of IgM ELISA in acute leptospirosis. Many samples were positive for both IgG and IgM antibodies. Polymerase Chain Reaction (PCR) targeting lipl41 gene was standardized and urine and blood samples from the same dogs were tested. PCR was found to be the specific test for the early detection of leptospires in blood even before seroconversion. However, PCR analysis of the urine samples was found to be insensitive. Hence, it can be concluded that the diagnostic strategies should be modified, and a combination of serological and molecular tests is recommended in endemic areas rather than simple detection of IgM or IgG antibodies, for the early detection of acute clinical cases of leptospirosis.
    Matched MeSH terms: Recombinant Proteins/immunology
  7. Fulltext Using transgenic plants and modified plant viruses for the development of treatments for human diseases
    Loh HS, Green BJ, Yusibov V
    Curr Opin Virol, 2017 10;26:81-89.
    PMID: 28800551 DOI: 10.1016/j.coviro.2017.07.019
    Production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. A major milestone for plant-based protein production for use in human health was achieved when Protalix BioTherapeutics produced taliglucerase alfa (Elelyso®) in suspension cultures of a transgenic carrot cell line for the treatment of patients with Gaucher's disease, was approved by the USA Food and Drug Administration in 2012. In this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism*
  8. Fulltext Use of FabV-Triclosan Plasmid Selection System for Efficient Expression and Production of Recombinant Proteins in Escherichia coli
    Ali SA, Chew YW, Omar TC, Azman N
    PLoS One, 2015;10(12):e0144189.
    PMID: 26642325 DOI: 10.1371/journal.pone.0144189
    Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.
    Matched MeSH terms: Recombinant Proteins/biosynthesis*; Recombinant Proteins/genetics
  9. Unscrambling the effect of C-terminal tail deletion on the stability of a cold-adapted, organic solvent stable lipase from Staphylococcus epidermidis AT2
    Kamarudin NH, Rahman RN, Ali MS, Leow TC, Basri M, Salleh AB
    Mol Biotechnol, 2014 Aug;56(8):747-57.
    PMID: 24771007 DOI: 10.1007/s12033-014-9753-1
    Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile-Thr-Arg-Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein-solvent interaction.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  10. Fulltext Turoctocog alfa pegol provides effective management for major and minor surgical procedures in patients across all age groups with severe haemophilia A: Full data set from the pathfinder 3 and 5 phase III trials
    Tosetto A, Neff A, Lentz SR, Santagostino E, Nemes L, Sathar J, et al.
    Haemophilia, 2020 May;26(3):450-458.
    PMID: 32293786 DOI: 10.1111/hae.13980
    INTRODUCTION: Turoctocog alfa pegol is a glycoPEGylated recombinant factor VIII (FVIII) with an extended half-life developed for prophylaxis, treatment of bleeds and perioperative management in patients with haemophilia A.

    AIM: Evaluate the efficacy and safety of turoctocog alfa pegol treatment for major and minor surgeries in the pathfinder 3 and 5 phase III trials.

    METHODS: Adults/adolescents aged ≥12 years with severe haemophilia A (FVIII <1%) received perioperative turoctocog alfa pegol treatment planned to achieve FVIII activity levels >80% during major surgery (pathfinder 3). The primary end point was haemostatic efficacy during surgery; secondary end points were blood loss, haemostatic effect postsurgery, consumption, transfusions, safety and health economics. Children (0-11 years) undergoing minor surgeries received 20-75 IU/kg turoctocog alfa pegol at Investigator's discretion (pathfinder 5).

    RESULTS: pathfinder 3 included 35 patients undergoing 49 major surgeries. Haemostasis was successful in 47/49 (95.9%) surgeries; two had moderate haemostatic responses. Median (mean) blood loss during major surgery was 75 (322.6) mL. Four bleeds were reported postsurgery; three were successfully treated with turoctocog alfa pegol (one was not evaluated). On the day of surgery, overall mean (median) dose was 75.5 (74.5) IU/kg and mean (median) number of doses was 1.7 (2.0). Five procedures required 11 transfusions on the day of surgery or days 1-6. No safety concerns or inhibitors were identified. Forty-five minor surgeries in 23 children were performed without complications.

    CONCLUSION: Turoctocog alfa pegol was effective for perioperative haemostatic management of major and minor surgeries in patients across age groups with severe haemophilia A.

    Matched MeSH terms: Recombinant Proteins/pharmacology; Recombinant Proteins/therapeutic use
  11. Treatment of chronic hepatitis B infection using interferon
    Guan R
    Med J Malaysia, 2005 Jul;60 Suppl B:28-33.
    PMID: 16108170
    Four to 6 months of conventional interferon alpha (IFN-alpha) (5MU daily or 10MU three times weekly) resulted in HBeAg loss in approximately 33% of HBeAg positive patients (controls: 12%). Longer treatment duration improved HBeAg seroconversion. Children with chronic HBV infection and high ALT respond to IFN-a at similar rates. Good end-of-treatment (ET) biochemical and virological response were also achieved with IFN-alpha in HBeAg negative, HBV-DNA positive hepatitis patients. Sustained response (SR) however, was disappointing, but improved with longer duration of treatment: (10-15% SR with 4/6 months treatment: 30% SR with 24 months treatment). Weekly pegylated IFN-alpha2a (PegIFN-alpha2a) for 24 weeks gave a significantly higher HBeAg conversion rate (33%) than conventional IFN-alpha2a (25%). Fifty-two weeks of PegIFN-alpha2b gave a sustained HBeAg loss in 35% patients and HBeAg seroconversion in 29% patients. Similar results were obtained with 48 weeks of weekly PegIFN-alpha2a. PegIFN-alpha2a monotherapy was found to be superior to lamivudine monotherapy in affecting a 6-month SR (normal ALTs and HBV DNA < 20,000 copies/mL) in HBeAg negative/anti-HBe positive chronic hepatitis B patients. There is a tendency for IFN-a and lamivudine combination to result in better sustained response than lamivudine monotherapy. This tendency is also observed with PegIFN-a and lamivudine combination although the combination did not appear to be better than PegIFN-alpha monotherapy. IFN induced HBeAg seroconversion is durable, could increase over time and resulted in better overall survival and survival free of hepatic decompensation or hepatocellular cancer. The main advantage of IFN-a therapy is that a course of finite duration may achieve sustained off-therapy response in a proportion of both HBeAg positive and HBeAg negative chronic hepatitis B patients. However, IFN treatment is usually associated with side-effects, especially flu-like symptoms, fatigue, neutropenia, thrombocytopenia and depression. These are usually tolerable but may require dose modification and premature cessation of treatment (5%). Interferon therapy induced hepatitis flares may lead to decompensation in patients with cirrhosis and can be dangerous in patients with decompensated liver function despite dose reduction.
    Matched MeSH terms: Recombinant Proteins
  12. Transcriptome analysis of Burkholderia pseudomallei T6SS identifies Hcp1 as a potential serodiagnostic marker
    Chieng S, Mohamed R, Nathan S
    Microb Pathog, 2015 Feb;79:47-56.
    PMID: 25616255 DOI: 10.1016/j.micpath.2015.01.006
    Burkholderia pseudomallei, the causative agent of melioidosis, is able to survive extreme environments and utilizes various virulence factors for survival and pathogenicity. To compete and survive within these different ecological niches, B. pseudomallei has evolved specialized pathways, including the Type VI secretion systems (T6SSs), that have a role in pathogenesis as well as interbacterial interactions. We examined the expression profile of B. pseudomallei T6SS six gene clusters during infection of U937 macrophage cells. T6SS-5 was robustly transcribed while the other five clusters were not significantly regulated proposing the utility of T6SS-5 as a potential biomarker of exposure to B. pseudomallei. Transcription of T6SS regulators VirAG and BprB was also not significant during infection when compared to bacteria grown in culture. Guided by these findings, three highly expressed T6SS genes, tssJ-4, hcp1 and tssE-5, were expressed as recombinant proteins and screened against melioidosis patient sera by western analysis and ELISA. Only Hcp1 was reactive by both types of analysis. The recombinant Hcp1 protein was further evaluated against a cohort of melioidosis patients (n = 32) and non-melioidosis individuals (n = 20) sera and the data clearly indicates a higher sensitivity (93.7%) and specificity (100%) for Hcp1 compared to bacterial lysate. The detection of anti-Hcp1 antibodies in patients' sera indicating the presence of B. pseudomallei highlights the potential of Hcp1 to be further developed as a serodiagnostic marker for melioidosis.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics
  13. Transactivator protein: An alternative for delivery of recombinant proteins for safer reprogramming of induced Pluripotent Stem Cell
    Nordin F, Ahmad RNR, Farzaneh F
    Virus Res, 2017 05 02;235:106-114.
    PMID: 28408207 DOI: 10.1016/j.virusres.2017.04.007
    Induced pluripotent stem cells (iPSC) are somatic cells reprogrammed to pluripotency by forced expression of pluripotency factors. These cells are shown to have the same pluripotent potential as embryonic stem cells (ESC) and considered as an alternative to the much controversial usage of ESC which involved human embryos. However, the traditional method in reprogramming cells into iPSC using genome-integrating retro- or lenti- viruses remains an obstacle for its application in clinical setting. Although numerous studies have been conducted for a safer DNA-based reprogramming, reprogramming of iPSC by genetic modifications may raise the possibility of malignant transformation and has been a major limitation for its usage in clinical applications. Therefore, there is a need for an alternative method to reprogram the cells without the use of gene editing and a much safer way to deliver transcription factors to induce pluripotency on target cells. Using protein transduction approach, a number of studies have demonstrated the generation of human iPSCs from human fibroblasts and mouse embryonic fibroblasts by direct delivery of reprogramming proteins. In this review, the definition and mechanism of HIV-TAT protein (a type of protein transduction domain) in delivering recombinant proteins, including the potential of protein-based delivery to induce iPSC were further discussed.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism*
  14. Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris
    Lau YL, Fong MY
    Exp Parasitol, 2008 Jul;119(3):373-8.
    PMID: 18457835 DOI: 10.1016/j.exppara.2008.03.016
    The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n=20), acute (IgM positive, IgG positive; n=20) and chronic (IgM negative, IgG positive; n=20) toxoplasmosis patients, and toxoplasmosis negative control patients (n=20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients' serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P<0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.
    Matched MeSH terms: Recombinant Proteins/immunology*; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  15. Tioman virus, a novel paramyxovirus isolated from fruit bats in Malaysia
    Chua KB, Wang LF, Lam SK, Crameri G, Yu M, Wise T, et al.
    Virology, 2001 May 10;283(2):215-29.
    PMID: 11336547
    A search for the natural host of Nipah virus has led to the isolation of a previously unknown member of the family Paramyxoviridae. Tioman virus (TiV) was isolated from the urine of fruit bats (Pteropus hypomelanus) found on the island of the same name off the eastern coast of peninsular Malaysia. An electron microscopic study of TiV-infected cells revealed spherical and pleomorphic-enveloped viral particles (100--500 nm in size) with a single fringe of embedded peplomers. Virus morphogenesis occurred at the plasma membrane of infected cells and morphological features of negative-stained ribonucleoprotein complexes were compatible with that of viruses in the family Paramyxoviridae. Serological studies revealed no cross-reactivity with antibodies against a number of known Paramyxoviridae members except for the newly described Menangle virus (MenV), isolated in Australia in 1997. Failure of PCR amplification using MenV-specific primers suggested that this new virus is related to but different from MenV. For molecular characterization of the virus, a cDNA subtraction strategy was employed to isolate virus-specific cDNA from virus-infected cells. Complete gene sequences for the nucleocapsid protein (N) and phosphoprotein (P/V) have been determined and recombinant N and V proteins produced in baculovirus. The recombinant N and V proteins reacted with porcine anti-MenV sera in Western blot, confirming the serological cross-reactivity observed during initial virus characterization. The lack of a C protein-coding region in the P/V gene, the creation of P mRNA by insertion of 2-G residues, and the results of phylogenetic analyses all indicated that TiV is a novel member of the genus Rubulavirus.
    Matched MeSH terms: Recombinant Proteins/immunology
  16. Fulltext Thermal stability engineering of Glomerella cingulata cutinase
    Chin IS, Abdul Murad AM, Mahadi NM, Nathan S, Abu Bakar FD
    Protein Eng. Des. Sel., 2013 May;26(5):369-75.
    PMID: 23468570 DOI: 10.1093/protein/gzt007
    Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  17. The workability of Escherichia coli BL21 (DE3) and Pseudomonas putida KT2440 expression platforms with autodisplayed cellulases: a comparison
    Obeng EM, Brossette T, Ongkudon CM, Budiman C, Maas R, Jose J
    Appl Microbiol Biotechnol, 2018 Jun;102(11):4829-4841.
    PMID: 29675801 DOI: 10.1007/s00253-018-8987-4
    This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; β-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and β-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and β-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics
  18. The use of response surface methodology for enhanced production of a thermostable bacterial lipase in a novel yeast system
    Abu ML, Mohammad R, Oslan SN, Salleh AB
    Prep Biochem Biotechnol, 2021;51(4):350-360.
    PMID: 32940138 DOI: 10.1080/10826068.2020.1818256
    A thermostable bacterial lipase from Geobacillus zalihae was expressed in a novel yeast Pichia sp. strain SO. The preliminary expression was too low and discourages industrial production. This study sought to investigate the optimum conditions for T1 lipase production in Pichia sp. strain SO. Seven medium conditions were investigated and optimized using Response Surface Methodology (RSM). Five responding conditions namely; temperature, inoculum size, incubation time, culture volume and agitation speed observed through Plackett-Burman Design (PBD) method had a significant effect on T1 lipase production. The medium conditions were optimized using Box-Behnken Design (BBD). Investigations reveal that the optimum conditions for T1 lipase production and Biomass concentration (OD600) were; Temperature 31.76 °C, incubation time 39.33 h, culture volume 132.19 mL, inoculum size 3.64%, and agitation speed of 288.2 rpm with a 95% PI low as; 12.41 U/mL and 95% PI high of 13.65 U/mL with an OD600 of; 95% PI low as; 19.62 and 95% PI high as; 22.62 as generated by the software was also validated. These predicted parameters were investigated experimentally and the experimental result for lipase activity observed was 13.72 U/mL with an OD600 of 24.5. At these optimum conditions, there was a 3-fold increase on T1 lipase activity. This study is the first to develop a statistical model for T1 lipase production and biomass concentration in Pichia sp. Strain SO. The optimized production of T1 lipase presents a choice for its industrial application.
    Matched MeSH terms: Recombinant Proteins/biosynthesis*
  19. Fulltext The use of dornase alfa in patients with COVID-19
    Ramachandram DS, Kow CS, Hasan SS
    J Cyst Fibros, 2023 May;22(3):580.
    PMID: 36966036 DOI: 10.1016/j.jcf.2023.03.010
    Matched MeSH terms: Recombinant Proteins/therapeutic use
  20. The potential correlation between patient-reported symptoms and the use of additional haemostatic medication for joint bleeding in haemophilia patients with inhibitors: a post hoc exploratory analysis of recombinant activated factor VII data from the ADEPT2 trial
    Lentz SR, Rangarajan S, Karim FA, Andersen PD, Arkhammar P, Rosu G, et al.
    Blood Coagul Fibrinolysis, 2017 Apr;28(3):224-229.
    PMID: 27427786 DOI: 10.1097/MBC.0000000000000584
    : Haemophilia treatment guidelines advocate early home-based treatment of acute bleeds. In the ADEPT2 trial, data were collected on the home treatment of bleeds with recombinant activated factor VII (rFVIIa) in haemophilia patients with inhibitors and self-reported bleeding-related symptoms. A total of 93% of all bleeds, and 91.5% of joint bleeds, were treated successfully with one to three doses of 90 μg/kg rFVIIa. However, some patients self-administered additional haemostatic medication (AHM) up to 48 h after the first rFVIIa treatment. The aim of this trial was to investigate the relationship between patient-reported symptoms, time to treatment initiation, and the use of AHM. A post hoc analysis was conducted on 177 joint bleeds and the patient-reported categorical symptoms of pain, swelling, mobility, tingling, and warmth, and the pain visual analogue scale (VAS) score. Analyses were descriptive and used logistic regression modelling. Complete symptom data were available for 141, 136, and 129 joint bleeds at 0 or 1, 3, and 6 h, respectively. Pain and pain VAS assessments were the best predictors of AHM use. Patients who self-administered AHM had higher mean pain VAS scores at each time point; both pain and pain VAS scores declined over time. Time to treatment initiation was an independent predictor for AHM use. Higher initial pain scores and longer time to treatment were the best predictors for administration of AHM. The observation that some patients chose to self-infuse in the face of declining levels of pain warrants further study to better understand the reasons behind patient decision-making.
    Matched MeSH terms: Recombinant Proteins/administration & dosage; Recombinant Proteins/therapeutic use
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