Displaying publications 81 - 100 of 362 in total

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  1. Process intensification of core streptavidin production through high-cell-density cultivation of recombinant E. coli and a temperature-based refolding method
    Chua LH, Tan SC, Liew MWO
    J Biotechnol, 2018 Jun 20;276-277:34-41.
    PMID: 29679607 DOI: 10.1016/j.jbiotec.2018.04.012
    An intensified process was developed that enables high level production of recombinant core streptavidin (cSAV), a non-glycosylated tetrameric protein utilised in a wide range of applications. A pH-stat fed-batch feeding strategy was employed to achieve high-cell-density and improve volumetric yield of cSAV which was expressed as inclusion bodies (IBs). The effect of induction at different cell densities (OD 20, 60 and 100) on volumetric and specific yield were then studied. Highest volumetric yield of cSAV (1550 mg L-1) was obtained from induction at OD 100 without significant reductions in specific yield. To recover active cSAV from IBs, the possibility of refolding using a temperature-based refolding method was investigated. Refolded cSAV obtained from temperature-based refolding were then compared against cSAV refolded with conventional dialysis and dilution methods using quantitative and qualitative metrics. The temperature-based refolding method was found to improve the yield of cSAV by 6-18% in comparison to conventional methods without compromising quality. Intensification was achieved by reductions in process volumes and a more concentrated product stream. Using the newly developed process, the volumetric yield of cSAV IBs was improved by thirty-six fold in comparison to low-cell-density shake flask cultivation, and 33% of cSAV can be recovered from IBs at 90% purity.
    Matched MeSH terms: Recombinant Proteins/analysis; Recombinant Proteins/genetics; Recombinant Proteins/metabolism*; Recombinant Proteins/chemistry
  2. Fulltext Cell entry of a host-targeting protein of oomycetes requires gp96
    Trusch F, Loebach L, Wawra S, Durward E, Wuensch A, Iberahim NA, et al.
    Nat Commun, 2018 06 14;9(1):2347.
    PMID: 29904064 DOI: 10.1038/s41467-018-04796-3
    The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants.
    Matched MeSH terms: Recombinant Proteins/chemistry
  3. 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
  4. Fulltext Identification and validation of a novel panel of Plasmodium knowlesi biomarkers of serological exposure
    Herman LS, Fornace K, Phelan J, Grigg MJ, Anstey NM, William T, et al.
    PLoS Negl Trop Dis, 2018 Jun;12(6):e0006457.
    PMID: 29902183 DOI: 10.1371/journal.pntd.0006457
    BACKGROUND: Plasmodium knowlesi is the most common cause of malaria in Malaysian Borneo, with reporting limited to clinical cases presenting to health facilities and scarce data on the true extent of transmission. Serological estimations of transmission have been used with other malaria species to garner information about epidemiological patterns. However, there are a distinct lack of suitable serosurveillance tools for this neglected disease.

    METHODOLOGY/PRINCIPAL FINDINGS: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1-91.3%) and identified the most predictive antibody responses.

    CONCLUSIONS/SIGNIFICANCE: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level.

    Matched MeSH terms: Recombinant Proteins/immunology
  5. 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
  6. Fulltext The Sequence-specific Peptide-binding Activity of the Protein Sulfide Isomerase AGR2 Directs Its Stable Binding to the Oncogenic Receptor EpCAM
    Mohtar MA, Hernychova L, O'Neill JR, Lawrence ML, Murray E, Vojtesek B, et al.
    Mol Cell Proteomics, 2018 04;17(4):737-763.
    PMID: 29339412 DOI: 10.1074/mcp.RA118.000573
    AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a specific peptide motif (TTIYY). A synthetic TTIYY-containing peptide column was used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a mutant peptide library. Screening the human proteome for proteins harboring this motif revealed an enrichment in transmembrane proteins and we focused on validating EpCAM as a potential AGR2-interacting protein. AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TLIYY motif and surrounding EpCAM's detergent binding site. These data define a dominant site on AGR2 that mediates its specific peptide-binding function. EpCAM forms a model client protein for AGR2 to study how an ER-resident chaperone can dock specifically to a peptide motif and regulate the trafficking a protein destined for the secretory pathway.
    Matched MeSH terms: Recombinant Proteins/metabolism
  7. Fulltext Engineering a stable CHO cell line for the expression of a MERS-coronavirus vaccine antigen
    Nyon MP, Du L, Tseng CK, Seid CA, Pollet J, Naceanceno KS, et al.
    Vaccine, 2018 03 27;36(14):1853-1862.
    PMID: 29496347 DOI: 10.1016/j.vaccine.2018.02.065
    Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377-588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/immunology
  8. Fulltext Efficacy and safety of intravenous recombinant tissue plasminogen activator in mild ischaemic stroke: a meta-analysis
    You S, Saxena A, Wang X, Tan W, Han Q, Cao Y, et al.
    Stroke Vasc Neurol, 2018 Mar;3(1):22-27.
    PMID: 29600004 DOI: 10.1136/svn-2017-000106
    The benefits and safety of intravenous recombinant tissue plasminogen activator (IV-tPA) for patients with mild ischaemic stroke (MIS) are still unclear. The objective of this meta-analysis was to evaluate the efficacy and safety of IV-tPA as treatment for patients with MIS. We performed a systematic literature search across MEDLINE, Embase, Central, Global Health and Cumulative Index to Nursing and Allied Health Literature (CINAHL), from inception to 10 November 2016, to identify all related studies. Where possible, data were pooled for meta-analysis with odds ratio (OR) and corresponding 95% confidence interval (CI) using the fixed-effects model. MIS was defined as having National Institutes of Health Stroke Scale score of ≤6. We included seven studies with a total of 1591 patients based on the prespecified inclusion and exclusion criteria. The meta-analysis indicated a high odds of excellent functional outcome based on the modified Rankin Scale or Oxfordshire Handicap Score 0-1 (OR=1.43; 95% CI 1.14 to 1.79; P=0.002, I2=35%) in patients treated with IV-tPA compared with those not treated with IV-tPA (74.8% vs 67.6%). There was a high risk of symptomatic intracranial haemorrhage (sICH) with IV-tPA treatment (OR=10.13; 95% CI 1.93 to 53.02; P=0.006, I2=0%) (1.9% vs 0.0%) but not mortality (OR=0.78; 95% CI 0.43 to 1.43; P=0.43, I2=0%) (2.4% vs 2.9%). Treatment with IV-tPA was associated with better functional outcome but not mortality among patients with MIS, although there was an increased risk of sICH. Randomised trials are warranted to confirm these findings.
    Matched MeSH terms: Recombinant Proteins/administration & dosage
  9. Fulltext Heterologous Expression of a Novel Drug Transporter from the Malaria Parasite Alters Resistance to Quinoline Antimalarials
    Tindall SM, Vallières C, Lakhani DH, Islahudin F, Ting KN, Avery SV
    Sci Rep, 2018 02 06;8(1):2464.
    PMID: 29410428 DOI: 10.1038/s41598-018-20816-0
    Antimalarial drug resistance hampers effective malaria treatment. Critical SNPs in a particular, putative amino acid transporter were recently linked to chloroquine (CQ) resistance in malaria parasites. Here, we show that this conserved protein (PF3D7_0629500 in Plasmodium falciparum; AAT1 in P. chabaudi) is a structural homologue of the yeast amino acid transporter Tat2p, which is known to mediate quinine uptake and toxicity. Heterologous expression of PF3D7_0629500 in yeast produced CQ hypersensitivity, coincident with increased CQ uptake. PF3D7_0629500-expressing cultures were also sensitized to related antimalarials; amodiaquine, mefloquine and particularly quinine. Drug sensitivity was reversed by introducing a SNP linked to CQ resistance in the parasite. Like Tat2p, PF3D7_0629500-dependent quinine hypersensitivity was suppressible with tryptophan, consistent with a common transport mechanism. A four-fold increase in quinine uptake by PF3D7_0629500 expressing cells was abolished by the resistance SNP. The parasite protein localised primarily to the yeast plasma membrane. Its expression varied between cells and this heterogeneity was used to show that high-expressing cell subpopulations were the most drug sensitive. The results reveal that the PF3D7_0629500 protein can determine the level of sensitivity to several major quinine-related antimalarials through an amino acid-inhibitable drug transport function. The potential clinical relevance is discussed.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism
  10. Fulltext Microtiter miniature shaken bioreactor system as a scale-down model for process development of production of therapeutic alpha-interferon2b by recombinant Escherichia coli
    Tan JS, Abbasiliasi S, Kadkhodaei S, Tam YJ, Tang TK, Lee YY, et al.
    BMC Microbiol, 2018 01 04;18(1):3.
    PMID: 29439680 DOI: 10.1186/s12866-017-1145-9
    BACKGROUND: Demand for high-throughput bioprocessing has dramatically increased especially in the biopharmaceutical industry because the technologies are of vital importance to process optimization and media development. This can be efficiently boosted by using microtiter plate (MTP) cultivation setup embedded into an automated liquid-handling system. The objective of this study was to establish an automated microscale method for upstream and downstream bioprocessing of α-IFN2b production by recombinant Escherichia coli. The extraction performance of α-IFN2b by osmotic shock using two different systems, automated microscale platform and manual extraction in MTP was compared.

    RESULTS: The amount of α-IFN2b extracted using automated microscale platform (49.2 μg/L) was comparable to manual osmotic shock method (48.8 μg/L), but the standard deviation was 2 times lower as compared to manual osmotic shock method. Fermentation parameters in MTP involving inoculum size, agitation speed, working volume and induction profiling revealed that the fermentation conditions for the highest production of α-IFN2b (85.5 μg/L) was attained at inoculum size of 8%, working volume of 40% and agitation speed of 1000 rpm with induction at 4 h after the inoculation.

    CONCLUSION: Although the findings at MTP scale did not show perfect scalable results as compared to shake flask culture, but microscale technique development would serve as a convenient and low-cost solution in process optimization for recombinant protein.

    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics
  11. The Effects of One Amino Acid Substitutions at the C-Terminal Region of Thermostable L2 Lipase by Computational and Experimental Approach
    Sani HA, Shariff FM, Rahman RNZRA, Leow TC, Salleh AB
    Mol Biotechnol, 2018 Jan;60(1):1-11.
    PMID: 29058211 DOI: 10.1007/s12033-017-0038-3
    The substitutions of the amino acid at the predetermined critical point at the C-terminal of L2 lipase may increase its thermostability and enzymatic activity, or even otherwise speed up the unfolding of the protein structure. The C-terminal of most proteins is often flexible and disordered. However, some protein functions are directly related to flexibility and play significant role in enzyme reaction. The critical point for mutation of L2 lipase structure was predicted at the position 385 of the L2 sequence, and the best three mutants were determined based on I-Mutant2.0 software. The best three mutants were S385E, S385I and S385V. The effects of the substitution of the amino acids at the critical point were analysed with molecular dynamics simulation by using Yet Another Scientific Artificial Reality Application software. The predicted mutant L2 lipases were found to have lower root mean square deviation value as compared to L2 lipase. It was indicated that all the three mutants had higher compactness in the structure, consequently enhanced the stability. Root mean square fluctuation analysis showed that the flexibility of L2 lipase was reduced by mutations. Purified S385E lipase had an optimum temperature of 80 °C in Tris-HCl pH 8. The highest enzymatic activity of purified S385E lipase was obtained at 80 °C temperature in Tris-HCl pH 8, while for L2 lipase it was at 70 °C in Glycine-NaOH pH 9. The thermal stability of S385V lipase was enhanced as compared to other protein since that the melting point (T m) value was at 85.96 °C. S385I lipase was more thermostable compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 h preincubation.
    Matched MeSH terms: Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Recombinant Proteins/chemistry
  12. Fulltext A Lateral Flow Rapid Test for Human Toxocariasis Developed Using Three Toxocara canis Recombinant Antigens
    Yunus MH, Tan Farrizam SN, Abdul Karim IZ, Noordin R
    Am J Trop Med Hyg, 2018 Jan;98(1):32-38.
    PMID: 29141740 DOI: 10.4269/ajtmh.17-0632
    Laboratory diagnosis of toxocariasis is still a challenge especially in developing endemic countries with polyparasitism. In this study, three Toxocara canis recombinant antigens, rTES-26, rTES-30, and rTES-120, were expressed and used to prepare lateral flow immunoglobulin G4 (IgG4) dipsticks. The concordance of the results of the rapid test (comprising three dipsticks) with a commercial IgG-enzyme-linked immunosorbent assay (ELISA) (Cypress Diagnostics, Belgium) was compared against the concordance of two other commercial IgG-ELISA kits (Bordier, Switzerland and NovaTec, Germany) with the Cypress kit. Using Toxocara-positive samples, the concordance of the dipstick dotted with rTES-26, rTES-30, and rTES-120 was 41.4% (12/29), 51.7% (15/29), and 72.4% (21/29), respectively. When positivity with any dipstick was considered as an overall positive rapid test result, the concordance with the Cypress kit was 93% (27/29). Meanwhile, when compared with the results of the Cypress kit, the concordance of IgG-ELISA from NovaTec and Bordier was 100% (29/29) and 89.7% (26/29), respectively. Specific IgG4 has been recognized as a marker of active infection for several helminthic diseases; therefore, the two non-concordant results of the rapid test when compared with the NovaTec IgG-ELISA kit may be from samples of people with non-active infection. All the three dipsticks showed 100% (50/50) concordance with the Cypress kit when tested with serum from individuals who were healthy and with other infections. In conclusion, the lateral flow rapid test is potentially a good, fast, and easy test for toxocariasis. Next, further validation studies and development of a test with the three antigens in one dipstick will be performed.
    Matched MeSH terms: Recombinant Proteins
  13. Expression of stable and active human DNA topoisomerase I in Pichia pastoris
    Chan MK, Lim SK, Miswan N, Chew AL, Noordin R, Khoo BY
    Protein Expr Purif, 2018 Jan;141:52-62.
    PMID: 28893606 DOI: 10.1016/j.pep.2017.09.003
    This study described the isolation of the coding region of human topoisomerase I (TopoI) from MDA-MB-231 and the expression of multiple copy recombinant genes in four Pichia pastoris strains. First, polymerase chain reaction (PCR)-amplification of the enzyme coding region was performed. The PCR fragment was cloned into pPICZ-α-A vector and sequenced. It was then transformed into X33, GS115, SMD1168H and KM71H strains of Pichia. PCR-screening for positive clones was performed, and estimation of multiple copy integrants in each Pichia strain was carried out using agar plates containing increasing concentrations of Zeocin(®). The selected clones of multiple copy recombinant genes were then induced for TopoI expression in shaker flasks. GS115 and SMD1168 were found to be better Pichia strains to accommodate the recombinant gene for the expression of TopoI extracellularly. However, the DNA relaxation activity revealed that only the target enzyme in the culture supernatants of GS115-pPICZ-α-A-TopoI exhibited consistent enzyme activity over the cultivation time-points. Active enzyme activity was inhibited by Camptothecin. The enzyme produced can be used for in-house gel-based DNA relaxation assay development in performing high throughput screening for target-specific growth inhibitors that display similar effect as the TopoI inhibitors. These inhibitors may contribute to the improvement of the treatment of cancer patients.
    Matched MeSH terms: Recombinant Proteins/analysis; Recombinant Proteins/genetics; Recombinant Proteins/metabolism*; Recombinant Proteins/chemistry
  14. Fulltext Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs
    Fischer K, Diederich S, Smith G, Reiche S, Pinho Dos Reis V, Stroh E, et al.
    PLoS One, 2018;13(4):e0194385.
    PMID: 29708971 DOI: 10.1371/journal.pone.0194385
    Hendra virus (HeV) and Nipah virus (NiV) belong to the genus Henipavirus in the family Paramyxoviridae. Henipavirus infections were first reported in the 1990's causing severe and often fatal outbreaks in domestic animals and humans in Southeast Asia and Australia. NiV infections were observed in humans in Bangladesh, India and in the first outbreak in Malaysia, where pigs were also infected. HeV infections occurred in horses in the North-Eastern regions of Australia, with singular transmission events to humans. Bats of the genus Pteropus have been identified as the reservoir hosts for henipaviruses. Molecular and serological indications for the presence of henipa-like viruses in African fruit bats, pigs and humans have been published recently. In our study, truncated forms of HeV and NiV attachment (G) proteins as well as the full-length NiV nucleocapsid (N) protein were expressed using different expression systems. Based on these recombinant proteins, Enzyme-linked Immunosorbent Assays (ELISA) were developed for the detection of HeV or NiV specific antibodies in porcine serum samples. We used the NiV N ELISA for initial serum screening considering the general reactivity against henipaviruses. The G protein based ELISAs enabled the differentiation between HeV and NiV infections, since as expected, the sera displayed higher reactivity with the respective homologous antigens. In the future, these assays will present valuable tools for serosurveillance of swine and possibly other livestock or wildlife species in affected areas. Such studies will help assessing the potential risk for human and animal health worldwide by elucidating the distribution of henipaviruses.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/immunology; Recombinant Proteins/isolation & purification
  15. 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
  16. 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
  17. Site-saturation mutagenesis of mutant L-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase
    Ismail A, Illias RM
    J Ind Microbiol Biotechnol, 2017 Dec;44(12):1627-1641.
    PMID: 28921081 DOI: 10.1007/s10295-017-1980-6
    The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
    Matched MeSH terms: Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/secretion
  18. Septic miscarriage with toxic shock syndrome and disseminated intravascular coagulation (DIC): The role of surgery, recombinant activated factor VII and intravenous immunoglobulin (IVIG)
    Tan LN, Mariappa G, Voon HY, Suharjono H
    Med J Malaysia, 2017 12;72(6):380-381.
    PMID: 29308782 MyJurnal
    Severe sepsis with multi-organ failure is associated with a high mortality rate. This case report highlights the challenges and modalities available in the management of a lady with refractory shock and disseminated intravascular coagulation (DIC) due to toxic shock syndrome (TSS) from genital tract sepsis. Early surgical intervention to remove the source of infection, the use of recombinant activated factor VII to treat intractable disseminated intravascular coagulation and intravenous immunoglobulin to neutralise the circulating exotoxins, have been employed and shown to drastically improve outcomes.
    Matched MeSH terms: Recombinant Proteins/administration & dosage
  19. Fulltext Optimization of physical conditions for the production of thermostable T1 lipase in Pichia guilliermondii strain SO using response surface methodology
    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
  20. 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*
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