Displaying publications 1 - 20 of 360 in total

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  1. Abd Rahman NH, Jaafar NR, Abdul Murad AM, Abu Bakar FD, Shamsul Annuar NA, Md Illias R
    Int J Biol Macromol, 2020 Sep 15;159:577-589.
    PMID: 32380107 DOI: 10.1016/j.ijbiomac.2020.04.262
    Short-chain fructooligosaccharides (scFOSs) can be produced from the levan hydrolysis using levanase. Levanase from Bacillus lehensis G1 (rlevblg1) is an enzyme that specifically converts levan to scFOSs. However, the use of free levanase presents a lack of stability and reusability, thus hindering the synthesis of scFOSs for continuous reactions. Here, CLEAs for rlevblg1 were prepared and characterized. Cross-linked levanase aggregates using glutaraldehyde (CLLAs-ga) and bovine albumin serum (CLLAs-ga-bsa) showed the best activity recovery of 92.8% and 121.2%, respectively. The optimum temperature of CLLAs-ga and CLLAs-ga-bsa was increased to 35 °C and 40 °C, respectively, from its free rlevblg1 (30 °C). At high temperature (50 °C), the half-life of CLLAs-ga-bsa was higher than that of free rlevblg1 and CLLAs-ga. Both CLLAs exhibited higher stability at pH 9 and pH 10. Hyperactivation of CLLAs-ga-bsa was achieved with an effectiveness factor of more than 1 and with improved catalytic efficiency. After 3 h reaction, CLLAs-ga-bsa produced the highest total scFOSs yield of 35.4% and total sugar of 60.4% per gram levan. Finally, the reusability of CLLAs for 8 cycles with more than 50% activity retained makes them as a potential synthetic catalyst to be explored for scFOSs synthesis.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  2. Abd Wahid MA, Megat Mohd Noor MJ, Goto M, Sugiura N, Othman N, Zakaria Z, et al.
    Biosci Biotechnol Biochem, 2017 Aug;81(8):1642-1649.
    PMID: 28585494 DOI: 10.1080/09168451.2017.1329617
    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/pharmacology; Recombinant Proteins/chemistry
  3. Abd-Aziz N, Tan BC, Rejab NA, Othman RY, Khalid N
    Mol Biotechnol, 2020 Apr;62(4):240-251.
    PMID: 32108286 DOI: 10.1007/s12033-020-00242-2
    In the past decade, interest in the production of recombinant pharmaceutical proteins in plants has tremendously progressed because plants do not harbor mammalian viruses, are economically competitive, easily scalable, and capable of carrying out complex post-translational modifications required for recombinant pharmaceutical proteins. Mucuna bracteata is an essential perennial cover crop species widely planted as an underground cover in oil palm and rubber plantations. As a legume, they have high biomass, thrive in its habitat, and can fix nitrogen. Thus, M. bracteata is a cost-efficient crop that shows ideal characteristics as a platform for mass production of recombinant protein. In this study, we established a new platform for the transient production of a recombinant protein in M. bracteata via vacuum-assisted agro-infiltration. Five-week-old M. bracteata plants were vacuum infiltrated with Agrobacterium tumefaciens harboring a plasmid that encodes for an anti-toxoplasma immunoglobulin (IgG) under different parameters, including trifoliate leaf positional effects, days to harvest post-infiltration, and the Agrobacterium strain used. Our results showed that vacuum infiltration of M. bracteata plant with A. tumefaciens strain GV3101 produced the highest concentration of heterologous protein in its bottom trifoliate leaf at 2 days post-infiltration. The purified anti-toxoplasma IgG was then analyzed using Western blot and ELISA. It was demonstrated that, while structural heterogeneity existed in the purified anti-toxoplasma IgG from M. bracteata, its transient expression level was two-fold higher than the model platform, Nicotiana benthamiana. This study has laid the foundation towards establishing M. bracteata as a potential platform for the production of recombinant pharmaceutical protein.
    Matched MeSH terms: Recombinant Proteins/metabolism; Recombinant Proteins/pharmacology
  4. Abdo AIK, Ngoh YY, Lew MH, Dass SA, Rahumatullah A, Noordin R, et al.
    Biotechnol Appl Biochem, 2022 Feb;69(1):70-76.
    PMID: 33258152 DOI: 10.1002/bab.2082
    Lymphatic filariasis is a neglected parasitic disease that affects millions in tropical and subtropical countries and is caused by Wuchereria and Brugia species. Specific and sensitive detection methods are essential in mapping infected areas where rapid tests are needed to cover underdeveloped and remote regions, which facilitates eliminating the disease as a public health problem. A few commercialized rapid tests based on antigen or antibody detection are available, but the former only detects infection by Wuchereria species and cross-reacts with nonlymphatic filaria, whereas antibody detection might provide positive results of previous infection. Here, we report the production of three different recombinant immunoglobulin gamma (IgG)1 antibodies based on scFvs previously generated via human antibody phage display technology, that is, anti-BmR1 clone 4, anti-BmXSP clone 5B, and anti-BmXSP clone 2H2. The scFv sequences were cloned into a pCMV-IgG1 vector, then transfected into a HEK293F cell line. The generated antibodies were found to be able to bind to their respective targets even at relatively low concentration. Conjugation of Fc to scFv induces binder stability and provides multiple labeling sites for probes and signaling molecules that can be used in rapid tests.
    Matched MeSH terms: Recombinant Proteins
  5. Abdul Wahab R, Basri M, Raja Abdul Rahman RN, Salleh AB, Abdul Rahman MB, Leow TC
    Enzyme Microb Technol, 2016 Nov;93-94:174-181.
    PMID: 27702478 DOI: 10.1016/j.enzmictec.2016.08.020
    Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  6. Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MN, Ali AM, Mohd Lila MA
    Appl Biochem Biotechnol, 2008 Mar;144(3):249-61.
    PMID: 18556814
    Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.
    Matched MeSH terms: Recombinant Proteins/analysis; Recombinant Proteins/biosynthesis*; Recombinant Proteins/genetics; Recombinant Proteins/pharmacology*
  7. Abdullah JM, Mutum SS, Nasuha NA, Biswal BM, Ariff AR
    Neurol. Med. Chir. (Tokyo), 2002 Jun;42(6):259-63.
    PMID: 12116532
    A 28-year-old Malay man presented with progressive paraparesis over a period of 6 months. Magnetic resonance imaging of the spine revealed a thoracic intramedullary spinal cord tumor at the T-7 level with homogeneous enhancement following intravenous gadolinium administration. Laminectomy and partial decompression of the tumor was performed. Histological examination of the tumor revealed features of spindle cell hemangioendothelioma. The patient was managed with limited field radiotherapy followed by systemic interferon therapy. Good neurological improvement was seen subsequently. The patient has survived 48 months with growth restraint at the primary site, although residual neurological deficit persists. Immunotherapy should be considered as a treatment modality for intramedullary hemangioendothelioma of the spinal cord after surgery and radiotherapy.
    Matched MeSH terms: Recombinant Proteins
  8. Abiri R, Valdiani A, Maziah M, Shaharuddin NA, Sahebi M, Yusof ZN, et al.
    Curr Issues Mol Biol, 2016;18:21-42.
    PMID: 25944541
    Using transgenic plants for the production of high-value recombinant proteins for industrial and clinical applications has become a promising alternative to using conventional bioproduction systems, such as bacteria, yeast, and cultured insect and animal cells. This novel system offers several advantages over conventional systems in terms of safety, scale, cost-effectiveness, and the ease of distribution and storage. Currently, plant systems are being utilised as recombinant bio-factories for the expression of various proteins, including potential vaccines and pharmaceuticals, through employing several adaptations of recombinant processes and utilizing the most suitable tools and strategies. The level of protein expression is a critical factor in plant molecular farming, and this level fluctuates according to the plant species and the organs involved. The production of recombinant native and engineered proteins is a complicated procedure that requires an inter- and multi-disciplinary effort involving a wide variety of scientific and technological disciplines, ranging from basic biotechnology, biochemistry, and cell biology to advanced production systems. This review considers important plant resources, affecting factors, and the recombinant-protein expression techniques relevant to the plant molecular farming process.
    Matched MeSH terms: Recombinant Proteins/biosynthesis
  9. Abu ML, Nooh HM, Oslan SN, Salleh AB
    BMC Biotechnol, 2017 Nov 10;17(1):78.
    PMID: 29126403 DOI: 10.1186/s12896-017-0397-7
    BACKGROUND: Pichia guilliermondii was found capable of expressing the recombinant thermostable lipase without methanol under the control of methanol dependent alcohol oxidase 1 promoter (AOXp 1). In this study, statistical approaches were employed for the screening and optimisation of physical conditions for T1 lipase production in P. guilliermondii.

    RESULT: The screening of six physical conditions by Plackett-Burman Design has identified pH, inoculum size and incubation time as exerting significant effects on lipase production. These three conditions were further optimised using, Box-Behnken Design of Response Surface Methodology, which predicted an optimum medium comprising pH 6, 24 h incubation time and 2% inoculum size. T1 lipase activity of 2.0 U/mL was produced with a biomass of OD600 23.0.

    CONCLUSION: The process of using RSM for optimisation yielded a 3-fold increase of T1 lipase over medium before optimisation. Therefore, this result has proven that T1 lipase can be produced at a higher yield in P. guilliermondii.

    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism*; Recombinant Proteins/chemistry
  10. 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*
  11. Abubakar MB, Aini I, Omar AR, Hair-Bejo M
    J Biomed Biotechnol, 2011;2011:414198.
    PMID: 21541235 DOI: 10.1155/2011/414198
    Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).
    Matched MeSH terms: Recombinant Proteins/metabolism
  12. Ahmed IM, Khairani-Bejo S, Hassan L, Bahaman AR, Omar AR
    BMC Vet Res, 2015;11:275.
    PMID: 26530141 DOI: 10.1186/s12917-015-0587-2
    Brucella melitensis is the most important pathogenic species of Brucella spp. which affects goats and sheep and causes caprine and ovine brucellosis, respectively. Serological tests for diagnosis of brucellosis such as Rose Bengal plate test (RBPT) and enzyme-linked immunosorbent assay (ELISA) usually utilize smooth lipopolysaccharides (S-LPS) as a diagnostic antigen which could give false positive serological reactions. Outer membrane proteins (OMP) of B. melitensis have been used as alternative diagnostic antigens rather than S-LPS for differential serological diagnosis of brucellosis, mainly in ELISA with single recombinant OMP (rOMP) as a diagnostic antigen. Nevertheless, the use of single format mainly showed lack of sensitivity against the desired rOMP. Therefore, this study aimed to determine whether a newly developed rOMPs indirect ELISA (rOMPs I-ELISA), based on combination of rOMP25, rOMP28 and rOMP31of B. melitensis, has a potential benefit for use in the serodiagnosis of brucellosis.
    Matched MeSH terms: Recombinant Proteins
  13. 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
  14. 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
  15. Ali MS, Ganasen M, Rahman RN, Chor AL, Salleh AB, Basri M
    Protein J, 2013 Apr;32(4):317-25.
    PMID: 23645400 DOI: 10.1007/s10930-013-9488-z
    A new strain of psychrophilic bacteria (designated strain AMS8) from Antarctic soil was screened for extracellular lipolytic activity and further analyzed using molecular approach. Analysis of 16S rDNA showed that strain AMS8 was similar to Pseudomonas sp. A lipase gene named lipAMS8 was successfully isolated from strain AMS8, cloned, sequenced and overexpressed in Escherichia coli. Sequence analysis revealed that lipAMS8 consist of 1,431 bp nucleotides that encoded a polypeptide consisting of 476 amino acids. It lacked an N-terminal signal peptide and contained a glycine- and aspartate-rich nonapeptide sequence at the C-terminus, which are known to be the characteristics of repeats-in-toxin bacterial lipases. Furthermore, the substrate binding site of lipAMS8 was identified as S(207), D(255) and H(313), based on homology modeling and multiple sequence alignment. Crude lipase exhibited maximum activity at 20 °C and retained almost 50 % of its activity at 10 °C. The molecular weight of lipAMS8 was estimated to be 50 kDa via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal expression level was attained using the recombinant plasmid pET32b/BL21(DE3) expressed at 15 °C for 8 h, induced by 0.1 mM isopropyl β-D thiogalactoside (IPTG) at E. coli growth optimal density of 0.5.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  16. 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
  17. Alias FL, Nezhad NG, Normi YM, Ali MSM, Budiman C, Leow TC
    Mol Biotechnol, 2023 Nov;65(11):1737-1749.
    PMID: 36971996 DOI: 10.1007/s12033-023-00725-y
    Heterologous functional expression of the recombinant lipases is typically a bottleneck due to the expression in the insoluble fraction as inclusion bodies (IBs) which are in inactive form. Due to the importance of lipases in various industrial applications, many investigations have been conducted to discover suitable approaches to obtain functional lipase or increase the expressed yield in the soluble fraction. The utilization of the appropriate prokaryotic and eukaryotic expression systems, along with the suitable vectors, promoters, and tags, has been recognized as a practical approach. One of the most powerful strategies to produce bioactive lipases is using the molecular chaperones co-expressed along with the target protein's genes into the expression host to produce the lipase in soluble fraction as a bioactive form. The refolding of expressed lipase from IBs (inactive) is another practical strategy which is usually carried out through chemical and physical methods. Based on recent investigations, the current review simultaneously highlights strategies to express the bioactive lipases and recover the bioactive lipases from the IBs in insoluble form.
    Matched MeSH terms: Recombinant Proteins/chemistry
  18. 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
  19. Amerizadeh A, Khoo BY, Teh AY, Golkar M, Abdul Karim IZ, Osman S, et al.
    BMC Infect Dis, 2013;13:287.
    PMID: 23800344 DOI: 10.1186/1471-2334-13-287
    Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology; Recombinant Proteins/metabolism
  20. 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
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