Displaying publications 1 - 20 of 30 in total

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  1. Ng CL, Reaz MB
    Sensors (Basel), 2017 Mar 12;17(3).
    PMID: 28287493 DOI: 10.3390/s17030574
    Capacitive biosensors are an emerging technology revolutionizing wearable sensing systems and personal healthcare devices. They are capable of continuously measuring bioelectrical signals from the human body while utilizing textiles as an insulator. Different textile types have their own unique properties that alter skin-electrode capacitance and the performance of capacitive biosensors. This paper aims to identify the best textile insulator to be used with capacitive biosensors by analysing the characteristics of 6 types of common textile materials (cotton, linen, rayon, nylon, polyester, and PVC-textile) while evaluating their impact on the performance of a capacitive biosensor. A textile-insulated capacitive (TEX-C) biosensor was developed and validated on 3 subjects. Experimental results revealed that higher skin-electrode capacitance of a TEX-C biosensor yields a lower noise floor and better signal quality. Natural fabric such as cotton and linen were the two best insulating materials to integrate with a capacitive biosensor. They yielded the lowest noise floor of 2 mV and achieved consistent electromyography (EMG) signals measurements throughout the performance test.
  2. Goh HH, Ng CL, Loke KK
    Adv Exp Med Biol, 2018 11 2;1102:11-30.
    PMID: 30382566 DOI: 10.1007/978-3-319-98758-3_2
    Functional genomics encompasses diverse disciplines in molecular biology and bioinformatics to comprehend the blueprint, regulation, and expression of genetic elements that define the physiology of an organism. The deluge of sequencing data in the postgenomics era has demanded the involvement of computer scientists and mathematicians to create algorithms, analytical software, and databases for the storage, curation, and analysis of biological big data. In this chapter, we discuss on the concept of functional genomics in the context of systems biology and provide examples of its application in human genetic disease studies, molecular crop improvement, and metagenomics for antibiotic discovery. An overview of transcriptomics workflow and experimental considerations is also introduced. Lastly, we present an in-house case study of transcriptomics analysis of an aromatic herbal plant to understand the effect of elicitation on the biosynthesis of volatile organic compounds.
  3. Arul M, Roslani AC, Ng CL, Cheah SH
    Cytotechnology, 2014 May;66(3):481-91.
    PMID: 23824584 DOI: 10.1007/s10616-013-9600-4
    There is increasing evidence that a tumour comprises of heterogeneous population of cells. Thus, studying homogenous cell lines in vitro may yield results that are not reflective of the true situation in a tumour and studying low passage cell lines maintained in a heterogeneous population before they transform away from the original state may provide a more complete picture of colorectal cancer. A method was developed to isolate and establish low passage colorectal cancer cell lines from tumour biopsies. The media contents, combination of antimicrobials and specimen collection and transport conditions employed, successfully eliminated microbial contamination which is frequently present in samples obtained from the gastrointestinal tract. A variety of growth forms indicating a heterogeneous mixture of cells was seen in the initial cultures. Using fluorescence immunocytochemistry, primary tumour cultures were shown to variably express selected tumour markers, carcinoembryonic antigen and C2 antigen. These low passage cell lines growing in a heterogeneous environment would more closely reflect the characteristics of the cells of the original tumour.
  4. Kahar UM, Ng CL, Chan KG, Goh KM
    Appl Microbiol Biotechnol, 2016 Jul;100(14):6291-307.
    PMID: 27000839 DOI: 10.1007/s00253-016-7451-6
    Type I pullulanases are enzymes that specifically hydrolyse α-1,6 linkages in polysaccharides. This study reports the analyses of a novel type I pullulanase (PulASK) from Anoxybacillus sp. SK3-4. Purified PulASK (molecular mass of 80 kDa) was stable at pH 5.0-6.0 and was most active at pH 6.0. The optimum temperature for PulASK was 60 °C, and the enzyme was reasonably stable at this temperature. Pullulan was the preferred substrate for PulASK, with 89.90 % adsorbance efficiency (various other starches, 56.26-72.93 % efficiency). Similar to other type I pullulanases, maltotriose was formed on digestion of pullulan by PulASK. PulASK also reacted with β-limit dextrin, a sugar rich in short branches, and formed maltotriose, maltotetraose and maltopentaose. Nevertheless, PulASK was found to preferably debranch long branches at α-1,6 glycosidic bonds of starch, producing amylose, linear or branched oligosaccharides, but was nonreactive against short branches; thus, no reducing sugars were detected. This is surprising as all currently known type I pullulanases produce reducing sugars (predominantly maltotriose) on digesting starch. The closest homologue of PulASK (95 % identity) is a type I pullulanase from Anoxybacillus sp. LM14-2 (Pul-LM14-2), which is capable of forming reducing sugars from starch. With rational design, amino acids 362-370 of PulASK were replaced with the corresponding sequence of Pul-LM14-2. The mutant enzyme formed reducing sugars on digesting starch. Thus, we identified a novel motif involved in substrate specificity in type I pullulanases. Our characterization may pave the way for the industrial application of this unique enzyme.
  5. Hashim OH, Ng CL, Gendeh S, Nik Jaafar MI
    Mol Immunol, 1991 4 1;28(4-5):393-8.
    PMID: 2062319
    The discovery of jacalin, a group of lectins from jackfruit seeds (Artocarpus heterophyllus), has attracted considerable attention due to its numerous interesting immunological properties as well as its usefulness in the isolation of various serum proteins. We have further identified a similar lectin from the seeds of Champedak (Artocarpus integer) which we refer to as lectin-C and performed comparative studies with two types of jacalin isolated from different batches of the Malaysian jackfruit seeds (jacalin-M1 and jacalin-M2). The three purified lectins demonstrated equivalent apparent Mr of about 52,500, each of which comprised of a combination of two types of non-covalently-linked subunits with apparent Mr of approximately 13,300 and 16,000. The lectins demonstrated equal haemagglutinating activity against human erythrocytes of blood groups A, B, AB and O. Our data also demonstrated that lectin-C, jacalin-M1 and jacalin-M2 are similar by selectively precipitating human serum IgA1 and colostral sIgA but not IgA2, IgD, IgG and IgM. When immunoelectrophoresis was performed on normal human sera and reacted with the lectins, single precipitin arcs corresponding to IgA immunoprecipitates were detected with lectin-C and jacalin-MI. Jacalin-M2, however, exhibited two closely associated precipitin arcs. The binding of these lectins with IgA was pronouncedly inhibited in the presence of p-nitrophenyl-beta-D-galactopyranoside, 1-o-methyl-alpha-D-galactopyranoside, D-melibiose, N-acetyl-D-galactosamine and D-galactose. The data therefore provide evidence on the differential specificity of IgA binding lectins isolated from seeds of similar as well as distinct Artocarpus species.
  6. Seman-Kamarulzaman AF, Mohamed-Hussein ZA, Ng CL, Hassan M
    PLoS One, 2016;11(8):e0161707.
    PMID: 27560927 DOI: 10.1371/journal.pone.0161707
    Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant.
  7. Shaibullah S, Mohd-Sharif N, Ho KL, Firdaus-Raih M, Nathan S, Mohamed R, et al.
    Acta Crystallogr F Struct Biol Commun, 2014 Dec 1;70(Pt 12):1697-700.
    PMID: 25484229 DOI: 10.1107/S2053230X14025278
    Melioidosis is an infectious disease caused by the pathogenic bacterium Burkholderia pseudomallei. Whole-genome sequencing revealed that the B. pseudomallei genome includes 5855 coding DNA sequences (CDSs), of which ∼25% encode hypothetical proteins. A pathogen-associated hypothetical protein, BPSL1038, was overexpressed in Escherichia coli, purified and crystallized using vapour-diffusion methods. A BPSL1038 protein crystal that grew using sodium formate as precipitant diffracted to 1.55 Å resolution. It belonged to space group C2221, with unit-cell parameters a = 85.36, b = 115.63, c = 46.73 Å. The calculated Matthews coefficient (VM) suggests that there are two molecules per asymmetric unit, with a solvent content of 48.8%.
  8. Krishnamurithy G, Shilpa PN, Ahmad RE, Sulaiman S, Ng CL, Kamarul T
    J Biomed Mater Res A, 2011 Dec 01;99(3):500-6.
    PMID: 21913317 DOI: 10.1002/jbm.a.33184
    Human amniotic membrane (HAM) is an established biomaterial used in many clinical applications. However, its use for tissue engineering purposes has not been fully realized. A study was therefore conducted to evaluate the feasibility of using HAM as a chondrocyte substrate/carrier. HAMs were obtained from fresh human placenta and were process to produced air dried HAM (AdHAM) and freeze dried HAM (FdHAM). Rabbit chondrocytes were isolated and expanded in vitro and seeded onto these preparations. Cell proliferation, GAG expression and GAG/cell expression were measured at days 3, 6, 9, 12, 15, 21, and 28. These were compared to chondrocytes seeded onto plastic surfaces. Histological analysis and scanning electron microscopy was performed to observe cell attachment. There was significantly higher cell proliferation rates observed between AdHAM (13-51%, P=0.001) or FdHAM (18-48%, p = 0.001) to chondrocytes in monolayer. Similarly, GAG and GAG/cell expressed in AdHAM (33-82%, p = 0.001; 22-60%, p = 0.001) or FdHAM (41-81%, p = 0.001: 28-60%, p = 0.001) were significantly higher than monolayer cultures. However, no significant differences were observed in the proliferation rates (p = 0.576), GAG expression (p = 0.476) and GAG/cell expression (p = 0.135) between AdHAM and FdHAM. The histology and scanning electron microscopy assessments demonstrates good chondrocyte attachments on both HAMs. In conclusion, both AdHAM and FdHAM provide superior chondrocyte proliferation, GAG expression, and attachment than monolayer cultures making it a potential substrate/carrier for cell based cartilage therapy and transplantation.
  9. Pang SL, Ho KL, Waterman J, Teh AH, Chew FT, Ng CL
    Acta Crystallogr F Struct Biol Commun, 2015 Nov;71(Pt 11):1396-400.
    PMID: 26527267 DOI: 10.1107/S2053230X1501818X
    Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.
  10. Chia JY, Tan WS, Ng CL, Hu NJ, Foo HL, Ho KL
    Sci Rep, 2016 08 09;6:31210.
    PMID: 27502833 DOI: 10.1038/srep31210
    DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed.
  11. Remali J, Loke KK, Ng CL, Aizat WM, Tiong J, Zin NM
    Genom Data, 2017 Sep;13:7-10.
    PMID: 28580299 DOI: 10.1016/j.gdata.2017.05.015
    Streptomyces sp. produces bioactive compounds with a broad spectrum of activities. Streptomyces kebangsaanesis SUK12 has been identified as a novel endophytic bacteria isolated from ethnomedicinal plant Portulaca olerace, and was found to produce the phenazine class of biologically active antimicrobial metabolites. The potential use of the phenazines has led to our research interest in determining the genome sequence of Streptomyces kebangsaanensis SUK12. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number PRJNA269542. The raw sequence data are available [https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP105770].
  12. Satyaveanthan MV, Suhaimi SA, Ng CL, Muhd-Noor ND, Awang A, Lam KW, et al.
    Plant Physiol Biochem, 2021 Apr;161:143-155.
    PMID: 33588320 DOI: 10.1016/j.plaphy.2021.01.050
    The juvenile hormones (JH) in plants are suggested to act as a form of plant defensive strategy especially against insect herbivory. The oxidation of farnesol to farnesoic acid is a key step in the juvenile hormone biosynthesis pathway. We herein present the purification and characterisation of the recombinant Theobroma cacao farnesol dehydrogenase enzyme that catalyses oxidation of farnesol to farnesal. The recombinant enzyme was purified to apparent homogeneity by affinity chromatography. The purified enzyme was characterised in terms of its deduced amino acid sequences, phylogeny, substrate specificity, kinetic parameters, structural modeling, and docking simulation. The phylogenetic analysis indicated that the T. cacao farnesol dehydrogenase (TcFolDH) showed a close relationship with A. thaliana farnesol dehydrogenase gene. The TcFolDH monomer had a large N-terminal domain which adopted a typical Rossmann-fold, harboring the GxxGxG motif (NADP(H)-binding domain) and a small C-terminal domain. The enzyme was a homotrimer comprised of subunits with molecular masses of 36 kDa. The TcFolDH was highly specific to NADP+ as coenzyme. The substrate specificity studies showed trans, trans-farnesol was the most preferred substrate for the TcFolDH, suggesting that the purified enzyme was a NADP+-dependent farnesol dehydrogenase. The docking of trans, trans-farnesol and NADP+ into the active site of the enzyme showed the important residues, and their interactions involved in the substrate and coenzyme binding of TcFolDH. Considering the extensive involvement of JH in both insects and plants, an in-depth knowledge on the recombinant production of intermediate enzymes of the JH biosynthesis pathway could help provide a potential method for insect control.
  13. Ker DS, Chan KG, Othman R, Hassan M, Ng CL
    Phytochemistry, 2020 May;173:112286.
    PMID: 32059132 DOI: 10.1016/j.phytochem.2020.112286
    The chemical formation of terpenes in nature is carried out by terpene synthases as the main biocatalysts to guide the carbocation intermediate to form structurally diverse compounds including acyclic, mono- and multiple cyclic products. Despite intensive study of the enzyme active site, the mechanism of specific terpene biosynthesis remains unclear. Here we demonstrate that a single mutation of the amino acid L454G or L454A in the active site of Persicaria minor β-sesquiphellandrene synthase leads to a more promiscuous enzyme that is capable of producing additional hydroxylated sesquiterpenes such as sesquicineole, sesquisabinene hydrate and α-bisabolol. Furthermore, the same L454 residue mutation (L454G or L454A) in the active site also improves the protein homogeneity compared to the wild type protein. Taken together, our results demonstrate that residue Leucine 454 in the active site of β-sesquiphellandrene synthase is important for sesquiterpene product diversity as well as the protein homogeneity in solution.
  14. Remali J, Aizat WM, Ng CL, Lim YC, Mohamed-Hussein ZA, Fazry S
    PeerJ, 2020;8:e9197.
    PMID: 32509463 DOI: 10.7717/peerj.9197
    Background: DNA double strand break repair is important to preserve the fidelity of our genetic makeup after DNA damage. Rad50 is one of the components in MRN complex important for DNA repair mechanism. Rad50 mutations can lead to microcephaly, mental retardation and growth retardation in human. However, Rad50 mutations in human and other organisms have never been gathered and heuristically compared for their deleterious effects. It is important to assess the conserved region in Rad50 and its homolog to identify vital mutations that can affect functions of the protein.

    Method: In this study, Rad50 mutations were retrieved from SNPeffect 4.0 database and literature. Each of the mutations was analyzed using various bioinformatic analyses such as PredictSNP, MutPred, SNPeffect 4.0, I-Mutant and MuPro to identify its impact on molecular mechanism, biological function and protein stability, respectively.

    Results: We identified 103 mostly occurred mutations in the Rad50 protein domains and motifs, which only 42 mutations were classified as most deleterious. These mutations are mainly situated at the specific motifs such as Walker A, Q-loop, Walker B, D-loop and signature motif of the Rad50 protein. Some of these mutations were predicted to negatively affect several important functional sites that play important roles in DNA repair mechanism and cell cycle signaling pathway, highlighting Rad50 crucial role in this process. Interestingly, mutations located at non-conserved regions were predicted to have neutral/non-damaging effects, in contrast with previous experimental studies that showed deleterious effects. This suggests that software used in this study may have limitations in predicting mutations in non-conserved regions, implying further improvement in their algorithm is needed. In conclusion, this study reveals the priority of acid substitution associated with the genetic disorders. This finding highlights the vital roles of certain residues such as K42E, C681A/S, CC684R/S, S1202R, E1232Q and D1238N/A located in Rad50 conserved regions, which can be considered for a more targeted future studies.

  15. Tan CS, Hassan M, Mohamed Hussein ZA, Ismail I, Ho KL, Ng CL, et al.
    Plant Physiol Biochem, 2018 Feb;123:359-368.
    PMID: 29304481 DOI: 10.1016/j.plaphy.2017.12.033
    Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp280, Leu294 and Ala303). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily.
  16. Pang SL, Matta SA, Sio YY, Ng YT, Say YH, Ng CL, et al.
    Sci Rep, 2021 01 13;11(1):921.
    PMID: 33441720 DOI: 10.1038/s41598-020-79820-y
    House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P 
  17. Ng CL, Reaz MBI, Crespo ML, Cicuttin A, Chowdhury MEH
    Sci Rep, 2020 09 10;10(1):14891.
    PMID: 32913303 DOI: 10.1038/s41598-020-71709-0
    A capacitive electromyography (cEMG) biomedical sensor measures the EMG signal from human body through capacitive coupling methodology. It has the flexibility to be insulated by different types of materials. Each type of insulator will yield a unique skin-electrode capacitance which determine the performance of a cEMG biomedical sensor. Most of the insulator being explored are solid and non-breathable which cause perspiration in a long-term EMG measurement process. This research aims to explore the porous medical bandages such as micropore, gauze, and crepe bandage to be used as an insulator of a cEMG biomedical sensor. These materials are breathable and hypoallergenic. Their unique properties and characteristics have been reviewed respectively. A 50 Hz digital notch filter was developed and implemented in the EMG measurement system design to further enhance the performance of these porous medical bandage insulated cEMG biomedical sensors. A series of experimental verifications such as noise floor characterization, EMG signals measurement, and performance correlation were done on all these sensors. The micropore insulated cEMG biomedical sensor yielded the lowest noise floor amplitude of 2.44 mV and achieved the highest correlation coefficient result in comparison with the EMG signals captured by the conventional wet contact electrode.
  18. Selvaraj BA, Mariatulqabtiah AR, Ho KL, Ng CL, Yong CY, Tan WS
    Int J Mol Sci, 2021 Aug 13;22(16).
    PMID: 34445426 DOI: 10.3390/ijms22168725
    The causative agent of white tail disease (WTD) in the giant freshwater prawn is Macrobrachium rosenbergii nodavirus (MrNV). The recombinant capsid protein (CP) of MrNV was previously expressed in Escherichia coli, and it self-assembled into icosahedral virus-like particles (VLPs) with a diameter of approximately 30 nm. Extensive studies on the MrNV CP VLPs have attracted widespread attention in their potential applications as biological nano-containers for targeted drug delivery and antigen display scaffolds for vaccine developments. Despite their advantageous features, the recombinant MrNV CP VLPs produced in E. coli are seriously affected by protease degradations, which significantly affect the yield and stability of the VLPs. Therefore, the aim of this study is to enhance the stability of MrNV CP by modulating the protease degradation activity. Edman degradation amino acid sequencing revealed that the proteolytic cleavage occurred at arginine 26 of the MrNV CP. The potential proteases responsible for the degradation were predicted in silico using the Peptidecutter, Expasy. To circumvent proteolysis, specific protease inhibitors (PMSF, AEBSF and E-64) were tested to reduce the degradation rates. Modulation of proteolytic activity demonstrated that a cysteine protease was responsible for the MrNV CP degradation. The addition of E-64, a cysteine protease inhibitor, remarkably improved the yield of MrNV CP by 2.3-fold compared to the control. This innovative approach generates an economical method to improve the scalability of MrNV CP VLPs using individual protease inhibitors, enabling the protein to retain their structural integrity and stability for prominent downstream applications including drug delivery and vaccine development.
  19. Ker DS, Sha HX, Jonet MA, Hwang JS, Ng CL
    Sci Rep, 2021 Oct 19;11(1):20649.
    PMID: 34667248 DOI: 10.1038/s41598-021-99879-5
    Actinoporins are a family of α-pore-forming toxins (α-PFTs) that have been identified in sea anemones. Recently, a freshwater Hydra Actinoporin-Like Toxin (HALT) gene family was found in Hydra magnipapillata. Unlike sea anemone actinoporins that use sphingomyelin as their main recognition target, the HALTs proteins may recognise alternative lipid molecules as their target. To unveil the structural insights into lipid preference of HALTs protein as compared to sea anemone actinoporins, we have determined the first crystal structure of actinoporin-like toxin, HALT-1 at 1.43 Å resolution with an acetylated lysine residue K76. Despite the overall structure of HALT-1 sharing a high structural similarity to sea anemone actinoporins, the atomic resolution structure revealed several unique structural features of HALT-1 that may influence the lipid preference and oligomerisation interface. The HALT-1 contains a RAG motif in place of the highly conserved RGD motif found in sea anemone actinoporins. The RAG motif contributed to a sharper β9-β10 turn, which may sway its oligomerisation interface in comparison to sea anemone actinoporins. In the lipid-binding region, the HALT-1 contains a shorter α2 helix and a longer α2-β9 loop due to deletion and subsequently an insertion of five amino acid residues in comparison to the sea anemone actinoporins. Structure comparison and molecular docking analysis further revealed that the HALT-1 lipid-binding site may favour sphingolipids with sulfate or phosphate head group more than the sphingomyelin. The structure of HALT-1 reported here provides a new insight for a better understanding of the evolution and lipid recognition mechanism of actinoporin.
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